Mouse Lymphoma Research Articles

KATP channels contribute to NK cell cytotoxicity.

ATP-sensitive K+ (KATP) channels regulate diverse processes (e.g. insulin secretion from the pancreas and blood flow) but they may also have a role in immunity. Knockout of Kcnj8 (Kir6.1) in mice leads to an exaggerated susceptibility to lipopolysaccharide (LPS). Moreover, a random mutagenesis screen in mice identified the mayday mutation (both Kcnj8 exons deleted) with a profound susceptibility to infection by mouse cytomegalovirus (MCMV) and a ∼20,000-fold sensitization to LPS, poly(I.C) and CpG DNA. Natural killer (NK) cells are effector lymphocytes of the innate immune system that mediate anti-tumor and microbial responses. Despite having key role in immune defenses, their complement of ion channels and how these channels regulate their function are largely uncharacterized compared to other immune cells. We postulated that KATP channels affect NK cell function. Analysis of microarray and RNA-seq mouse ImmGen datasets shows that, compared to other immune cells, Kcnj8 mRNA is expressed selectively and at high levels in natural killer (NK) cells and in CD8+ effector memory T cells after viral infection. We confirmed that Kir6.1 protein is expressed in isolated mouse splenic NK cells. Expansion of mouse splenic NK cells with IL-15 and IL-2 leads to elevated levels of the cytotoxic granzyme and perforin proteins, which is further increased in the presence of the KATP channel opener pinacidil (30 µM). Co-culture of mouse lymphoma YAC-1 cells with mouse splenic NK cells leads to apoptosis progression of the target cells, as well as increased degranulation of NK cells assessed by surface expression of CD107a assessed by flow cytometry. Apoptosis progression and NK cell degranulation were both significantly enhanced by pinacidil (1-30 µM). These data are consistent with a role for KATP channels in NK cell cytotoxicity, possibly by contributing to the release of cytotoxic proteins from NK cells.

The In Vivo Intervention and Relative Mechanism of Genistein on the Inflammation and Thrombophilia in Lymphoma-Bearing Mice

To investigate the in vivo intervention and relative mechanism of Genistein (GEN) on tumor-associated inflammatory and tumor thrombophilia in lymphoma-bearing mice. Forty female Balb/c mice aged 5-6 weeks were injected with murine-derived Pro B-cell lymphoma cell line 38B9 to establish a lymphoma mouse model, which was randomly divided into control group, tumor-bearing group, GEN drug intervention group and cyclophosphamide (CTX）drug intervention group. Histopathologic was used to evaluate the tumorigenesis. Tumor formation was observed, and tumor tissues were collected of HE and immunohistochemical staining. ELISA and flow cytometry were used to detect the expression of inflammatory factors and the changes of thrombus indices in plasma after intervention of GEN and Cyclophosphamide (CTX) respectively. Immunohistochemistry method was used to detect the expression of CD19 in tomor tissues of tummor bearing mice. After 14 days of tumor bearing, the mice were tumorigenic. The lymphoma cells were diffusely distributed in the tumor tissue and the expression of CD19 in the tumor tissue was positive. The inflammatory factors such as IL-6, NETs and CLEC-2, and thrombotic indices such as TF, FIB and D-D in lymphoma-bearing mice were significantly higher than those before tumor-injection and lower than those after drug-intervention (all P<0.05). The levels of CLEC-2 and D-D in GEN group were significantly lower than those in CTX group (P<0.05). Tumor-associated inflammation and thrombophilia exist in lymphoma-bearing mice. GEN shows better anti-inflammatory and anti-thrombotic effects compared with CTX by interfering with tumor inflammatory factors.

Enhanced antitumor effect of curcumin loaded solid lipid nanoparticles in Dalton’s ascites lymphoma mice

Curcumin is widely known for its antibacterial, antioxidant and anti inflammatory effects and has been reported to possess anticancerous activity as well. However, its medical application is limited because of poor bioavailability and rapid metabolism. In this study, we encapsulated curcumin in solid lipid nanoparticles and studied its anticancerous effect in Dalton’s Ascites Lymphoma (DAL) mice model. The physicochemical characteristics of curcumin solid lipid nanoparticles (CUR-SLN) were assessed and the anticancer efficacy was determined by in vivo studies. The curcumin solid lipid nanoparticles were synthesized by solvent emulsification evaporation method with particle size less than 100 nm. Antitumor effect of nanocurcumin (50 mg/kg) and curcumin (100 mg/kg) was evaluated in Dalton’s Ascites Lymphoma bearing mice. Pathological and immunohistochemical parameters were studied. Mean survival time and percentage increase in lifespan were assessed. Nanocurcumin group showed more significant influence in reducing tumor volume and weight, inducing apoptosis, reducing angiogenesis and invasion restoring antioxidant parameters and increased mean survival time. Curcumin and nanocurcumin inhibited the activation of nuclear factor-kappa B (Nf-kB), and thereby proved the pathway by which it induced anti-angiogenic and anti-invasive property.

Targeted Lymphoma Therapy Using a Gold Nanoframework-Based Drug Delivery System.

Precision nanomedicine can be employed as an alternative to chemo- or radiotherapy to overcome challenges associated with the often narrow therapeutic window of traditional treatment approaches, while safely inducing effective, targeted antitumor responses. Herein, we report the formulation of a therapeutic nanocomposite comprising a hyaluronic acid (HA)-coated gold nanoframework (AuNF) delivery system and encapsulated IT848, a small molecule with potent antilymphoma and -myeloma properties that targets the transcriptional activity of nuclear factor kappa B (NF-κB). The porous AuNFs fabricated via a liposome-templated approach were loaded with IT848 and surface-functionalized with HA to formulate the nanotherapeutics that were able to efficiently deliver the payload with high specificity to myeloma and lymphoma cell lines in vitro. In vivo studies characterized biodistribution, pharmacokinetics, and safety of HA-AuNFs, and we demonstrated superior efficacy of HA-AuNF-formulated IT848 vs free IT848 in lymphoma mouse models. Both in vitro and in vivo results affirm that the AuNF system can be adopted for targeted cancer therapy, improving the drug safety profile, and enhancing its efficacy with minimal dosing. HA-AuNF-formulated IT848 therefore has strong potential for clinical translation.

The altering cellular components and function in tumor microenvironment during remissive and relapsed stages of anti-CD19 CAR T-cell treated lymphoma mice.

Anti-CD19 chimeric antigen receptor (CAR) T cells represent a highly promising strategy for B-cell malignancies. Despite the inspiring initial achievement, remission in a notable fraction of subjects is short-lived, and relapse remains a major challenge. Tumor microenvironment (TME) was proved to be aroused by CAR T cells; however, little is known about the dynamic characteristics of cellular components in TME especially during the different phases of disease after anti-CD19 CAR T-cell treatment. We took advantage of an immunocompetent model receiving syngeneic A20 lymphoma cells to dissect the changes in TME with or without CAR T-cell injection. We found that anti-CD19 CAR T-cell treatment attenuated the symptoms of lymphoma and significantly prolonged mice survival through eradicating systemic CD19+ cells. Increased myeloid subsets, including CD11c+ DCs and F4/80+ macrophages with higher MHC II and CD80 expression in bone marrow, spleen, and liver, were detected when mice reached remission after anti-CD19 CAR T treatment. Compared to mice without anti-CD19 CAR T administration, intrinsic T cells were triggered to produce more IFN-γ and TNF-α. However, some lymphoma mice relapsed by day 42 after therapy, which coincided with CAR T-cell recession, decreased myeloid cell activation and increased Treg cells. Elevated intrinsic T cells with high PD-1 and TIGIT exhaust signatures and attenuated cytotoxicity in TME were associated with the late-stage relapse of CAR T-cell treatment. In summary, the cellular compositions of TME as allies of CAR T cells may contribute to the anti-tumor efficacy at the initial stage, whereas anti-CD19 CAR T-cell disappearance and host response immunosuppression may work together to cause lymphoma relapse after an initial, near-complete elimination phase.

Genotoxicity evaluation of graphene derivatives by a battery of in vitro assays

The interest of graphene materials has increased markedly in the recent years for their promising applications in many fields as food packing. These new applications have caused some concern regarding their safety for consumers since the intake of these materials may increase. In this sense, a battery of in vitro test is required before its use as a food contact material. Then, the aim of this study was to assess the potential mutagenicity and genotoxicity of graphene oxide (GO) and reduced-graphene oxide (rGO) following the recommendations of the European Food Safety Authority (EFSA). Thus, the mouse lymphoma assay (MLA) and the micronucleus test (MN) were performed in L5178YTk ± cells, and the Caco-2 cells were used for the standard and modified comet assays. The results indicated that GO (0–250 μg/mL) was not mutagenic in the MLA. However, rGO revealed mutagenic activity from 250 μg/mL and 125 μg/mL after 4h and 24h of exposure, respectively. In the MN test, negative results were obtained for both compounds at the concentrations assayed (0–250 μg/mL) for GO/rGO. Moreover, no DNA strand breaks, or oxidative DNA damage were detected in Caco-2 cells exposed to GO (0–250 μg/mL) and rGO (0–176.3 μg/mL for 24h and 0–166.5 μg/mL for 48h). Considering the mutagenic potential of rGO observed further investigation is needed to describe its toxic profile.

S1PR1/S1PR3-YAP signaling and S1P-ALOX15 signaling contribute to an aggressive behavior in obesity-lymphoma

BackgroundExcess body weight has been found to associate with an increased risk of lymphomas and some metabolic pathways are currently recognized in lymphomagenesis. Bioactive lipid metabolites such as sphingosine-1-phosphate (S1P) have been proposed to play an important role linking obesity and lymphomas. However, the underlying mechanism(s) of S1P signaling in obesity-lymphomagenesis have not been well addressed.MethodsThe gene expression of sphingosine kinase (SPHK), lymphoma prognosis, and S1P production were analyzed using Gene Expression Omnibus (GEO) and human lymphoma tissue array. Obesity-lymphoma mouse models and lymphoma cell lines were used to investigate the S1P/SPHK-YAP axis contributing to obesity-lymphomagenesis. By using the mouse models and a monocyte cell line, S1P-mediated polarization of macrophages in the tumor microenvironment were investigated.ResultsIn human study, up-regulated S1P/SPHK1 was found in human lymphomas, while obesity negatively impacted progression-free survival and overall survival in lymphoma patients. In animal study, obesity-lymphoma mice showed an aggressive tumor growth pattern. Both in vivo and in vitro data suggested the existence of S1P-YAP axis in lymphoma cells, while the S1P-ALOX15 signaling mediated macrophage polarization towards TAMs exacerbated the lymphomagenesis. In addition, treatment with resveratrol in obesity-lymphoma mice showed profound effects of anti-lymphomagenesis, via down-regulating S1P-YAP axis and modulating polarization of macrophages.ConclusionS1P/S1PR initiated the feedback loops, whereby S1P-S1PR1/S1PR3-YAP signaling mediated lymphomagenesis contributing to tumor aggressive growth, while S1P-ALOX15 signaling mediated TAMs contributing to immunosuppressive microenvironment in obesity-lymphoma. S1P-targeted therapy could be potentially effective and immune-enhancive against obesity-lymphomagenesis.Graphical

FOXP1-GINS1 axis promotes DLBCL proliferation and directs doxorubicin resistance.

GINS1 is overexpressed in several types of cancers including leukemia and linked to poor outcomes. However, GINS1 remains poorly investigated in DLBCL (diffuse large B-cell lymphoma). This project aimed to explore the expression, functions and regulation of GINS1 in DLBCL. In this study, through analysis of clinical specimens from DLBCL patients, we uncovered that GINS1 was upregulated in DLBCL. By EMSA, ChIP and luciferase reporter assays, it was found that FOXP1 transcriptionally activated GINS1 expression by directly binding to the promoter region of the GINS1 gene. Western blotting and RT-PCR also revealed that GINS1 expression positively correlated with FOXP1 in human DLBCL specimens and cell lines. In an in vivo xenograft lymphoma mouse model, the FOXP1/GINS1 regulatory axis was also validated. Moreover, with CCK8 cell proliferation assays and colony formation assay, elevated GINS1 expression was found to be associated with doxorubicin resistance in lymphoma cells. Our findings showed that the FOXP1-GINS1 axis played a critical role in DLBCL development and doxorubicin resistance, and targeting the FOXP1-GINS1 axis could be a potential therapeutic approach for DLBCL treatment.

Cysteine-specific 89Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas.

Positron emission tomography (PET) using radiolabeled Abs as imaging tracer is called immuno-PET. Immuno-PET can verify therapeutic Ab delivery and can noninvasively quantify global levels of target expression in tumors of living subjects. The interleukin-2 receptor α chain (IL-2Rα; CD25) is a promising target for immune therapy and radioimmunotherapy of lymphomas. Immuno-PET could facilitate this approach by visualizing CD25 expression in vivo. We prepared 89Zr-anti-CD25 IgG specifically labeled to sulfhydryl moieties by maleimide-deferoxamine conjugation. CD25(+) SUDHL1 human T-cell lymphoma cells showed high anti-human 89Zr-CD25 IgG binding that reached 32-fold of that of CD25(-) human lymphoma cells and was completely blocked by excess unlabeled Ab. In SUDHL1 tumor-bearing nude mice, pharmacokinetic studies demonstrated exponential reductions of whole blood and plasma activity following intravenous 89Zr-anti-CD25 IgG injection, with half-lives of 26.0 and 23.3 h, respectively. SUDHL1 tumor uptake of 89Zr-CD25 IgG was lower per weight in larger tumors, but blood activity did not correlate with tumor size or blood level of human CD25, indicating minimal influence by circulating soluble CD25 protein secreted from the lymphoma cells. 89Zr-CD25 IgG PET allowed high-contrast SUDHL1 lymphoma visualization at five days. Biodistribution studies confirmed high tumor 89Zr-CD25 IgG uptake (8.7 ± 0.9%ID/g) that was greater than blood (5.2 ± 1.6%ID/g) and organ uptakes (0.7 to 3.5%ID/g). Tumor CD25-specific targeting was confirmed by suppression of tumor uptake to 4.3 ± 0.2%ID by excess unlabeled CD25 IgG, as well as by low tumor uptake of 89Zr-labeled IgG2a isotype control Ab (3.6 ± 0.9%ID). Unlike CD25(+) lymphocytes from mouse thymus that showed specific uptake of anti-mouse 89Zr-CD25 IgG, EL4 mouse lymphoma cells had low CD25 expression and showed low uptake. In immunocompetent mice bearing EL4 tumors, anti-mouse 89Zr-CD25 IgG displayed low uptakes in normal organs as well as in the tumor. Furthermore, the biodistribution was not influenced by Ab blocking, indicating that specific uptake in nontumor tissues was minimal. 89Zr-CD25 IgG immuno-PET may thus be useful for imaging of T-cell lymphomas and noninvasive assessment of CD25 expression on target cells in vivo.

Skewed fate and hematopoiesis of CD34+ HSPCs in umbilical cord blood amid the COVID-19 pandemic.

Umbilical cord blood (UCB) is an irreplaceable source for hematopoietic stem progenitor cells (HSPCs). However, the effects of SARS-CoV-2 infection and COVID-19 vaccination on UCB phenotype, specifically the HSPCs therein, are currently unknown. We thus evaluated any effects of SARS-CoV-2 infection and/or COVID-19 vaccination from the mother on the fate and functionalities of HSPCs in the UCB. The numbers and frequencies of HSPCs in the UCB decreased significantly in donors with previous SARS-CoV-2 infection and more so with COVID-19 vaccination via the induction of apoptosis, likely mediated by IFN-γ-dependent pathways. Two independent hematopoiesis assays, a colony forming unit assay and a mouse humanization assay, revealed skewed hematopoiesis of HSPCs obtained from donors delivered from mothers with SARS-CoV-2 infection history. These results indicate that SARS-CoV-2 infection and COVID-19 vaccination impair the functionalities and survivability of HSPCs in the UCB, which would make unprecedented concerns on the future of HSPC-based therapies.

Effect of Special Structure Bi-Specific Chimeric Antigen Receptor T Cell on Tumor Cells

To explore and design a novel bi-specific chimeric antigen receptor (CAR) structure. To obtain the corresponding CAR-T cells and verify killing effects on tumor cells in vitro and in vivo. Five kinds of bi-specific CAR structures including humanized CD19 scFv and CD79b scFv, CD8 hinge & TM-4-1BB-CD3ζ and/or CD3ε chain intracellular regions were constructed and prepared. CAR-19-79b cells were obtained. Five kinds of CAR-T cells were co-incubated with the 3M-CD19-CD79b-Luc target cells. Luciferase assay and ELISA were used to detecte the killing ability of these five groups of CAR-T cells and the secretion of cytokines and compared. The optimal structure of CAR-T cells was used to treat the leukemia mouse model constructed by Daudi-Luc cells. And the treatment efficacy was evaluated. At the same time, other targets were used in this structure. With the same methods, the stability and effectiveness of the structure were verified. CAR-19-79b-T cells were cultured for 7 days, the expression rates of CAR-19 and CAR-79b were 21.6%-36.3% and 21.7%-37.8%, respectively. The killing rates of 5 kinds of CAR-19-79b-T cells prepared by T cells from 3 healthy donors on 3M-CD19-CD79b-Luc cells were significantly higher than those of the T cell control group at the effect-target ratio of 10∶1. Among them, the killing rates of CAR-19-79b-T cells with No. III and No. IV structures were the strongest. After co-incubation with 3M-CD19-CD79b-Luc target cells, the amount of IFN-γ and TNF-α secreted by CAR-T cells with CAR IV and CARV structures was the lowest. And there was no significance between the two groups (P>0.05). CAR IV cells with remarkable killing effect and low secretion factor had obvious therapeutic effect on Daudi-Luc leukemia mice, extending the survival period of mice to 64 days. And all mice in the T cell control group died at 41.0±2.4 days. The CAR-19-BCMA-T and CAR-19-22-T with the same structure showed significant killing ability and low cytokine expression levels. A novel bi-specific CAR structures was successfully designed, which could efficiently kill the corresponding tumor cells and secrete less cytokines (such as TNF-α, IFN-γ). Moreover, it shows obvious therapeutic effect on Daudi lymphoma mouse model. The bi-specific CAR structure shows good killing specificity and safety.

In vitro and in vivo assessments of the genotoxic potential of 3-chloroallyl alcohol.

3-Chloroallyl alcohol (3-CAA) can be found in the environment following the application of plant protection products. 3-CAA is formed in groundwater following the injection of 1,3-dichloropropene, a fumigant used to control nematodes. 3-CAA is also formed, in leafy crops, as a glycoside conjugate following application of the herbicide, clethodim. Human exposure may occur from groundwater used as drinking water or through dietary consumption. To characterize 3-CAA's potential to cause genotoxicity in mammals, in vitro and in vivo studies were conducted. 3-CAA was negative in an Ames test and positive in a mouse lymphoma forward mutation assay. 3-CAA was negative in an acute in vivo CD-1 mouse bone marrow micronucleus assay when administered up to a dose level of 125 mg/kg/day for two consecutive days. In a combined gene mutation assay and erythrocyte micronucleus assay, using transgenic Big Blue® Fischer 344 rats, 3-CAA was administered via drinking water at targeted dose levels of 0, 10, 30, and 100 mg/kg/day for 29 days. Peripheral blood samples, collected at the end of treatment, were analyzed for micronucleus induction in reticulocytes using flow cytometry. Liver and bone marrow samples, collected 2 days after the termination of the treatment, were analyzed for the induction of mutations at the cII locus. 3-CAA did not induce an increase in mutant frequency or micronuclei under the experimental conditions. In conclusion, the mutagenic response observed in the in vitro mouse lymphoma assay is not confirmed in the whole animal. 3-CAA is not considered to pose a mutagenic risk.

Targeted Nanotherapy of Hematologic Malignancies Using Gold Nanoframework-Based Delivery of a Novel NF-Kb Inhibitor

Hematologic malignancies account for one third of all cancers worldwide. Traditional chemo- or radiotherapy of these often highly aggressive cancers is limited by poor bio-selectivity and a narrow safety window, correlating with a risk for serious side effects and the development of refractory disease. Precision molecular therapeutics and highly targeted nanotechnological approaches can be employed as an alternative to overcome these challenges and successfully produce targeted anti-tumor responses, especially in the setting of disseminated disease. Herein, we report the formulation of a therapeutic nanocomposite comprising of a hyaluronic acid (HA)-coated gold nanoframework delivery system and encapsulated IT848, a novel small molecule with potent anti-lymphoma and -myeloma properties that targets the transcriptional activity of nuclear factor kappa B (NF-κB). We show that IT848 inhibits NF-κB activity through inhibition of DNA binding of all five NF-κB subunits. IT848 treatment of lymphoma and myeloma cell lines and patient samples inhibited proliferation and induced caspase-dependent and independent apoptosis. In addition to direct NF-κB inhibitory effects, IT848 treatment altered the redox homeostasis of cancer cells through depletion of the reduced glutathione pool, selectively inducing oxidative stress in cancer but not in healthy cells. IT848 significantly improved survival in an immunocompetent mouse model of multiple myeloma when combined with PD-1 inhibition, and correlative immune studies revealed that this clinical benefit was associated with suppression of regulatory T cell infiltration of the tumor microenvironment. We optimized the safety profile as well as the efficacy of IT848 by adapting HA-coated gold nanoparticles as an integrative platform for CD44-targeted delivery of IT848. Among a plethora of functionalized nanomaterials used for drug delivery, gold nanoparticles/nanoframeworks (AuNF) demonstrate excellent efficacy as drug carriers, photothermal or contrast agents, and radiosensitizers. Furthermore, easy synthesis and apt control over particle size, shape, porosity, and surface chemistry, together with their excellent biocompatibility make these particles ideal vehicles for small molecules such as IT848. HA-functionalized and IT848-loaded AuNFs were synthesized via a simple, reproducible, and inexpensive liposome-assisted technique that can easily be scaled up, a critical feature for clinical relevance. AuNFs were loaded with IT848 and surface functionalized with HA to formulate nanotherapeutics that were able to efficiently deliver the payload with high specificity to myeloma and lymphoma cell lines in vitro. In vivo studies characterized biodistribution, pharmacokinetics and safety of HA-AuNFs, and we demonstrated significantly superior specificity and efficacy of HA-AuNF formulated IT848 versus free IT848 in lymphoma mouse models (Figure 1). Our results indicate that active targeted delivery of IT848 using AuNFs via HA-CD44 ligand-receptor mediated endocytosis is a promising strategy to augment IT848 uptake by CD44-overexpressing tumor cells, mitigate rapid clearance of nanoparticles from the body and yield a highly desirable and sustained (as compared to free IT848) anti-tumor therapeutic effect. Our HA-AuNF-based drug delivery approach offers infrequent (weekly) low dose administration and robust efficacy with minimal toxicity risk. In fact, i.v. administration of IT848-loaded HA-AuNFs once or twice a week was not associated with any signs of toxicity, while traditional intraperitoneal administration of free IT848 twice a week over a 2-week period typically leads to ascites development in up to 50% of the recipients. Collectively, both our in vitro and in vivo results affirm that our AuNF system facilitates targeted drug delivery, improving the drug safety profile, and enhancing the efficacy of our novel NF-kB inhibitor molecule with minimal dosing. Our data therefore provide a strong preclinical rationale for the clinical development of HA-AuNF formulated IT848 as a therapeutic modality for the treatment of hematological malignancies. Additional studies are needed to develop curative treatment regimens based on synergistic, highly active, and well-tolerated combinatorial approaches with other targeted agents such as immune checkpoint inhibitors, hypomethylating agents, or CAR-T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SP-3164, a Novel Cereblon-Binding Protein Degrader, Shows Activity in Preclinical Lymphoma Models

Background: Targeted protein degradation (TPD) is attracting significant interest given the success of early protein degraders, e.g., lenalidomide and pomalidomide, and TPD's potential advantages over other therapeutic approaches. SP-3164, an oral, next-generation molecular glue, is currently in IND-enabling studies and is expected to enter the clinic in 2023. SP-3164 interacts with the cereblon (CRBN) component of a CRL4 E3 ligase, inducing recruitment of select proteins, i.e., Ikaros (IKZF1) and Aiolos (IKZF3), leading to their ubiquitination and proteasomal degradation. To date, most CRBN-binding compounds, including SP-3164, contain a chiral aminoglutarimide moiety and exist as (S)- and (R)-enantiomers. Studies have shown that in many of these CRBN-binding compounds, it is the (S)-enantiomer that binds to CRBN with increased affinity and thus may be the primary species resulting in the desired protein degradation and anti-cancer activity. However, the (S)- and (R)-enantiomers can chemically interconvert, preventing development of a single enantiomer. SP-3164 was designed using deuterium to stabilize the (S)-enantiomer with the potential for an improved therapeutic profile compared to the clinically studied racemate, avadomide. We previously demonstrated that compared to avadomide, SP-3164 shows increased binding affinity to CRBN, while the deuterium-stabilized (R)-enantiomer (SP-3165) does not bind CRBN at clinically relevant concentrations. Here, we further characterize SP-3164 pharmacological properties compared to avadomide, SP-3165, and lenalidomide, and demonstrate its activity in preclinical lymphoma models. Results: In addition to CRBN binding, efficiency of TPD is another critical parameter. In in vitro studies of diffuse large B cell lymphoma (DLBCL) cell lines of different subtypes (WSU-DLCL2, SU-DHL-5, SU-DHL-10 and U-2932), SP-3164 (500 nM) resulted in greater IKZF1 degradation at 6 hours (Figure 1A), when compared to avadomide (AVA, 1 µM) or lenalidomide (LEN, 10 µM). By contrast, SP-3165 (500 nM) did not result in meaningful IKZF1 depletion, further corroborating the (R)-enantiomer as the inactive species. Pharmacokinetics (PK) of SP-3164, SP-3165, and avadomide were studied in mice. Oral administration of either SP-3164 or SP-3165 resulted in increased exposure to the dosed enantiomer when compared to an equivalent dose of avadomide, and minimal exposure to the other enantiomer, while the elimination half-life of SP-3164 and SP-3165 was approximately half that of the enantiomers of avadomide. Therefore, SP-3164 has the potential to be administered at lower doses in clinic and provide a more flexible dosing regimen compared to avadomide, while still maintaining sufficient exposure for anticancer activity. The in vivo efficacy of SP-3164 for lymphoma was studied in a WSU-DLCL2 mouse xenograft model (Figure 1B). Compared to lenalidomide, SP-3164 exhibited significantly increased tumor growth inhibition (TGI). SP-3164 and avadomide showed similar activity at comparable doses and dosing regimens (SP-3164, 15 mg/kg QD, and avadomide, 30 mg/kg QD). SP-3165 lacked significant anti-cancer effects indicating that the (R)-enantiomer does not contribute to the direct anti-cancer activity of avadomide, as anticipated based on its lack of binding to CRBN. Due to the shorter elimination half-life of SP-3164 compared to avadomide, SP-3164 was also dosed BID. The SP-3164 BID treated group had the largest TGI, suggesting that the in vivo anti-cancer activity may be due to sustained exposure of SP-3164 at the tumor resulting in prolonged reduction of target protein levels. Conclusion: SP-3164 is a novel, CRBN-binding molecular glue with potential attractive therapeutic properties compared to the racemate, avadomide, and to lenalidomide. SP-3164 showed increased IKZF1 degradation efficiency compared to other molecular glues and significant anti-cancer activity as a monotherapy in an in vivo lymphoma mouse xenograft model. Future studies will evaluate SP-3164 efficacy and direct potential combinations to support clinical development of SP-3164 in DLBCL and other non-Hodgkin's lymphomas. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genotoxicity testing of nanomaterials.

Nanomaterials have outstanding and unprecedented advantageous material properties but may also cause adverse effects in humans upon exposure. Testing nanomaterials for genotoxic properties is challenging because traditional testing methods were designed for small, soluble molecules and may not be easily applicable without modifications. This review critically examines available genotoxicity tests for use with nanomaterials, including DNA damage tests such as the comet assay, gene mutation tests such as the mouse lymphoma and hprt assay, and chromosome mutation tests such as the micronucleus test and the chromosome aberration test. It presents arguments for the relative usefulness of various tests, such as preferring the micronucleus test over the chromosome aberration test for scoring chromosome mutations and preferring mammalian cell gene mutation tests because the Ames test has limited utility. Finally, it points out the open questions and further needs in adapting genotoxicity tests for nanomaterials, such as validation, reference nanomaterials, and the selection of top test concentrations, as well as the relevance and applicability of test systems and the need to define testing strategies. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Toxicological assessment of dihydroberberine.

A battery of studies was conducted to examine the toxicological potential of dihydroberberine (DHBBR), a derivative of berberine (BBR). The genotoxicity studies conducted on DHBBR, including the bacterial reverse mutation test, the mouse lymphoma assay, and the in vivo micronucleus test, showed that DHBBR is non-mutagenic and non-clastogenic. An acute oral toxicity study revealed that the LD50 of DHBBR in female Sprague Dawley rats was greater than 2000mg/kg bw. In a 14-day oral dose range finding study, the maximum tolerated dose was the high dose, 120mg/kgbw/day. Based on a 90-day oral toxicity study in male and female Sprague Dawley rats, it was concluded that the NOAEL for DHBBR is 100mg/kgbw/day, the highest dose tested.

Synthesis and single-molecule imaging reveal stereospecific enhancement of binding kinetics by the antitumour eEF1A antagonist SR-A3.

Ternatin-family cyclic peptides inhibit protein synthesis by targeting the eukaryotic elongation factor-1α. A potentially related cytotoxic natural product ('A3') was isolated from Aspergillus, but only 4 of its 11 stereocentres could be assigned. Here, we synthesized SR-A3 and SS-A3-two out of 128 possible A3 epimers-and discovered that synthetic SR-A3 is indistinguishable from naturally derived A3. Relative to SS-A3, SR-A3 exhibits an enhanced residence time and rebinding kinetics, as revealed by single-molecule fluorescence imaging of elongation reactions catalysed by eukaryotic elongation factor-1α in vitro. An increased residence time-stereospecifically conferred by the unique β-hydroxyl in SR-A3-was also observed in cells. Consistent with its prolonged duration of action, thrice-weekly dosing with SR-A3 led to a reduced tumour burden and increased survival in an aggressive Myc-driven mouse lymphoma model. Our results demonstrate the potential of SR-A3 as a cancer therapeutic and exemplify an evolutionary mechanism for enhancing cyclic peptide binding kinetics via stereospecific side-chain hydroxylation.

Co-expression and Interaction of Pax6 with Genes and Proteins of Immunological Surveillance in the Brain of Mice.

The Pax6 binds to promoter sequence elements of genes involved in immunological surveillance and interacts with Iba1, p53, Ras-GAP, and Sparc in the brain of mice. The Pax6 also affects the expression pattern of genes involved in neurogenesis and neurodegeneration. However, the expression and association of Pax6 in the brain under immunologically challenged conditions are still elusive. Therefore, it has been intended to analyze the association of Pax6 in the immunity of the brain using the immune-challenged Dalton's lymphoma (DL) mice model. The expressions of Pax6, Iba1, and Tmem119 decreased, but expressions of Ifn-γ, Tnf-α, Bdnf, and Tgf-β increased in the brain of immune-challenged mice as compared to the control. The level of co-expression of Pax6 decreased in dual positive cells with Iba1, Tmem119, Sparc, p53, Bdnf, and Tgf-β in the brain of immune-challenged mice. Binding of Pax6 to multiple sites of the promoter sequences of Bdnf and Tgf-β indicates their Pax6-associated differential expression and association with immune responsive gene. The levels of binding of Pax6 to Tmem119, Iba1, Ifn-γ, and Tnf-α got altered during the immune-challenged state as compared to control. Results provide the first evidence of the association of Pax6 in brain-specific immunity.

Abstract A26: SOX11 drives B1a lineage commitment and B1a/MCL-like lymphoma formation in mice

Abstract Mantle cell lymphoma (MCL) is a highly aggressive B cell lymphoma that accounts for 6% of all non-Hodgkin lymphomas and is characterized by abnormal proliferation of mature antigen- naive B lymphocytes. Despite recent advances in patient risk stratification, MCL remains incurable due to frequent relapses and resistance to therapy. SOX11 is a key transcription factor in the pathogenesis of MCL and is highly expressed in conventional MCL, but it is not expressed in the normal B cells or in the indolent leukemic nonnodal MCL subtype. Although SOX11 was identified as crucial oncogene that supports maintenance of MCL, its role in the initiation of MCL remains largely unknown. Here, we developed a novel mouse model for condition overexpression of SOX11 and a eGFP reporter and found that elevated levels of SOX11 in B-cells are sufficient to drive MCL-like lymphomas in mice. Moreover, SOX11 expression synergized with loss of p53 and overexpression of Ccnd2 to form aggressive MCL-like CD19+CD5+CD23−IgM+IgD+ lymphomas, with a median survival of 314 (p53Mb1 ; n=11), 165 (SOX11/p53Mb1 ; n=19), and 134 (SOX11/Ccnd2/p53Mb1 ; n=15) days. These SOX11-driven lymphomas show transcriptional, immunophenotypic and functional similarities with both murine B1a cells and MCL patients. SOX11 overexpression skews B-cell development towards the B1a and marginal zone B-cell lineage and it block B2-cell development at the Pro-B and Pre-B cell stage, which coincides with onset eGFP that is a measure of expression of Cre and SOX11. In contrast to cyclin D2-driven MCL-like model, SOX11 is inducing a pre-lymphoma stage, in which there is a progressive accumulation of B1a cells in peripheral blood, peritoneal cavity, spleen and bone-marrow. The pre-lymphoma B1a cells have a BCR repertoire which was biased towards binding of self-antigens, such as phosphatidylcholine, and have increased BCR signaling activity. To prove that embryonic-derived B1a cells were the cell-of-origin of SOX11-driven MCL-like lymphoma, we transplanted CD19+ B-cells from SOX11/p53Mb1 fetal livers into secondary recipients and found that these embryonic cells could give rise to MCL-like lymphomas. Next, we wanted to determine how SOX11 is inducing the pre-lymphoma phase, either by expanding existing B1a cells, having a developmental bias towards B1a cells, or by the ability to reprogram B2-cells towards a B1a cell fate. Therefore, we crossed SOX11/p53 mice with a tamoxifen-inducible CreERT2 line and transplanted different sorted B1 and B2 subsets after a short tamoxifen induction ex vivo in immunocompromised mice. We found that SOX11 OE can redirect mature B2 cells to form B1a/MCL lymphomas. Overall, SOX11 is a key B1a lineage factor in mice and its aberrant expression is leading to the formation of MCL-like lymphomas in mice. Citation Format: Tim Pieters, Steven Goossens, Pieter Van Vlierberghe. SOX11 drives B1a lineage commitment and B1a/MCL-like lymphoma formation in mice [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A26.

Size-dependent activation of CAR-T cells.

As the targets of chimeric antigen receptor (CAR)-T cells expand to a variety of cancers, autoimmune diseases, viral infections, and fibrosis, there is an increasing demand for identifying new antigens and designing new CARs that can be effectively activated. However, the rational selection of antigens and the design of CARs are limited by a lack of knowledge regarding the molecular mechanism by which CARs are activated by antigens. Here, we present data supporting a "size exclusion" model explaining how antigen signals are transmitted across the plasma membrane to activate the intracellular domains of CARs. In this model, antigen engagement with CAR results in a narrow intermembrane space that physically excludes CD45, a bulky phosphatase, out of the CAR zone, thus favoring CAR phosphorylation by kinases, which further triggers downstream pathways leading to T cell activation. Aligned with this model, increasing the size of CAR extracellular domains diminished CAR-T activation both in vitro and in a mouse lymphoma model; membrane-proximal epitopes activated CAR-Ts better than membrane-distal epitopes. Moreover, increasing the size of CD45 by antibody conjugation enhanced the activation of CARs that recognize membrane-distal epitopes. Consistently, CAR-Ts expressing CD45RABC, the larger isoform, were activated to a higher level than those expressing a smaller isoform CD45RO. Together, our work revealed that CAR-T activation depends on the size difference between the CAR-antigen pair and CD45; the size of CAR, antigen, and CD45 can thus be targets for tuning CAR-T activation.