Restricted Expression Pattern Research Articles

Cartography of teneurin and latrophilin expression reveals spatiotemporal axis heterogeneity in the mouse hippocampus during development.

Synaptic adhesion molecules (SAMs) are evolutionarily conserved proteins that play an important role in the form and function of neuronal synapses. Teneurins (Tenms) and latrophilins (Lphns) are well-known cell adhesion molecules that form a transsynaptic complex. Recent studies suggest that Tenm3 and Lphn2 (gene symbol Adgrl2) are involved in hippocampal circuit assembly via their topographical expression. However, it is not known whether other teneurins and latrophilins display similar topographically restricted expression patterns during embryonic and postnatal development. Here, we reveal the cartography of all teneurin (Tenm1-4) and latrophilin (Lphn1-3 [Adgrl1-3]) paralog expression in the mouse hippocampus across prenatal and postnatal development as monitored by large-scale single-molecule RNA in situ hybridization mapping. Our results identify a striking heterogeneity in teneurin and latrophilin expression along the spatiotemporal axis of the hippocampus. Tenm2 and Tenm4 expression levels peak at the neonatal stage when compared to Tenm1 and Tenm3, while Tenm1 expression is restricted to the postnatal pyramidal cell layer. Tenm4 expression in the dentate gyrus (DG) exhibits an opposing topographical expression pattern in the embryonic and neonatal hippocampus. Our findings were validated by analyses of multiple RNA-seq datasets at bulk, single-cell, and spatial levels. Thus, our study presents a comprehensive spatiotemporal map of Tenm and Lphn expression in the hippocampus, showcasing their diverse expression patterns across developmental stages in distinct spatial axes.

Excitatory Spinal Lhx9-Derived Interneurons Modulate Locomotor Frequency in Mice.

Locomotion allows us to move and interact with our surroundings. Spinal networks that control locomotion produce rhythm and left-right and flexor-extensor coordination. Several glutamatergic populations, Shox2 non-V2a, Hb9-derived interneurons, and, recently, spinocerebellar neurons have been proposed to be involved in the mouse rhythm generating networks. These cells make up only a smaller fraction of the excitatory cells in the ventral spinal cord. Here, we set out to identify additional populations of excitatory spinal neurons that may be involved in rhythm generation or other functions in the locomotor network. We use RNA sequencing from glutamatergic, non-glutamatergic, and Shox2 cells in the neonatal mice from both sexes followed by differential gene expression analyses. These analyses identified transcription factors that are highly expressed by glutamatergic spinal neurons and differentially expressed between Shox2 neurons and glutamatergic neurons. From this latter category, we identified the Lhx9-derived neurons as having a restricted spinal expression pattern with no Shox2 neuron overlap. They are purely glutamatergic and ipsilaterally projecting. Ablation of the glutamatergic transmission or acute inactivation of the neuronal activity of Lhx9-derived neurons leads to a decrease in the frequency of locomotor-like activity without change in coordination pattern. Optogenetic activation of Lhx9-derived neurons promotes locomotor-like activity and modulates the frequency of the locomotor activity. Calcium activities of Lhx9-derived neurons show strong left-right out-of-phase rhythmicity during locomotor-like activity. Our study identifies a distinct population of spinal excitatory neurons that regulates the frequency of locomotor output with a suggested role in rhythm-generation in the mouse alongside other spinal populations.

B-Glycine as a marker for β cell imaging and β cell mass evaluation.

β cell mass (BCM) and function are essential to the diagnosis and therapy of diabetes. Diabetic patients serve β cell loss is, and damage of β cells leads to severe insulin deficiency. Our understanding of the role of BCM in diabetes progression is extremely limited by lacking efficient methods to evaluate BCM in vivo. In vitro methods of labeling islets, including loading of contrast reagent or integration of exogenous biomarker, require artificial manipulation on islets, of which the clinical application is limited. Imaging methods targeting endogenous biomarkers may solve the above problems. However, traditional reagents targeting GLP-1R and VAMT2 result in a high background of adjacent tissues, complicating the identification of pancreatic signals. Here, we report a non-invasive and quantitative imaging technique by using radiolabeled glycine mimics ([18F]FBG, a boron-trifluoride derivative of glycine) to assay islet function and monitor BCM changes in living animals. Glycine derivatives, FBG, FBSa, 2Me-FBG, 3Me-FBG, were successfully synthesized and labeled with 18F. Specificity of glycine derivatives were characterized by in vitro experiment. PET imaging and biodistribution studies were performed in animal models carring GLYT over-expressed cells. In vivo evaluation of BCM with [18F]FBG were performed in STZ (streptozocin) induced T1D (type 1 diabetes) models. GLYT responds to excess blood glycine levels and transports glycine into islet cells to maintain the activity of the glycine receptor (GLYR). Best PET imaging condition was 80min after given a total of 240 ~ 250 nmol imaging reagent (a mixture of [18F]FBG and natural glycine) intravenously. [18F]FBG can detect both endogenous and exogenous islets clearly in vivo. When applied to STZ induced T1D mouse models, total uptake of [18F]FBG in the pancreas exhibited a linear correlation with survival BCM. [18F]FBG targeting the endogenous glycine transporter (GLYT), which is highly expressed on islet cells, avoiding extra modification on islet cells. Meanwhile the highly restricted expression pattern of GLYT excluded the background in adjacent tissues. This [18F]FBG-based imaging technique provides a non-invasive method to quantify BCM in vivo, implying a new evaluation index for diabetic assessment.

Regulation of hypothalamic reactive oxygen species and feeding behavior by phosphorylation of the beta 2 thyroid hormone receptor isoform

Unlike other thyroid hormone receptors (THRs), the beta 2 isoform (THRB2) has a restricted expression pattern and is uniquely and abundantly phosphorylated at a conserved serine residue S101 (S102 in humans). Using tagged and or phosphorylation-defective (S101A) THRB2 mutant mice, we show that THRB2 is present in a large subset of POMC neurons and mitigates ROS accumulation during ROS-triggering events, such as fasting/refeeding or high fat diet (HFD). Excessive ROS accumulation in mutant POMC neurons was accompanied by a skewed production of orexigenic/anorexigenic hormones, resulting in elevated food intake. The prolonged exposure to pathogenic hypothalamic ROS levels during HFD feeding lead to a significant loss of POMC neurons in mutant versus wild-type (WT) mice. In cultured cells, the presence of WT THRB2 isoform, but not other THRs, or THRB2S101A, reduced ROS accumulation upon exogenous induction of oxidative stress by tert-butyl hydroperoxide. The protective function of phospho-THRB2 (pTHRB2) did not require thyroid hormone (TH), suggesting a TH-independent role of the THRB2 isoform, and phospho-S101 in particular, in regulating oxidative stress. We propose that pTHRB2 has a fundamental role in neuronal protection against ROS cellular damage, and mitigates hypothalamic pathological changes found in diet-induced obesity.

Abstract 6727: QL335, a novel T cell engager with enhanced safety profile targeting MSS colorectal cancer

Abstract Colorectal cancer (CRC) is the fourth leading cause of death in the world with a global mortality rate of over 700,000 annually. While recent advancement in diagnosis and prevention has reduced the overall number of deaths, the lack of effective and durable treatment has not significantly improved survival rate. Emerging modalities in immunotherapy such as T cell engagers have paved new ways for disease management. However, a common challenge to advancing T cell engagers in the clinic is poor target specificity and high toxicity associated with non-specific T cell activation and cytokine release. To overcome these shortcomings, we have developed QL335, a bispecific antibody bridging LY6G6D antigen on tumor cells and CD3 on T cells. LY6G6D or Lymphocyte Antigen 6 Family Member G6D, is a GPI-anchored membrane protein that, according to TCGA RNAseq data, is highly expressed in microsatellite stable (MSS) colorectal cancer but not in normal tissues. We analyzed LY6G6D expression by IHC in over 200 CRC patient tumors and 40+ normal tissue samples from different organs, confirming its highly restricted CRC expression pattern. We constructed our bispecific antibody in a 2+1 format, comprising two high affinity LY6G6D binding domains and one reduced affinity CD3 binding domain. Such format allows for higher tumor accumulation while mitigating non-specific T cell activation in the periphery. In vitro evaluation showed that QL335 induced potent yet highly specific T cell activation and killing of only target cells expressing LY6G6D. QL335 demonstrated robust anti-tumor efficacy in vivo against human colon cancer xenograft models with human PBMC reconstitution. Importantly, QL335 was well tolerated in cynomolgus monkeys dosed up to 50 mg/kg, supporting the enhanced safety profile by design of this therapeutic candidate. QL335 is currently being evaluated in a phase 1 clinical trial. Citation Format: Shenda Gu, Guangjie Guo, Helena Yee, Hieu V. Tran, Shihao Chen, Chuanzeng Cao, Ke Liu, Chunyan Jin, Weihua Lun, Ruixue Ma, Lele Ren. QL335, a novel T cell engager with enhanced safety profile targeting MSS colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6727.

BAF45D-binding to HOX genes was differentially targeted in H9-derived spinal cord neural stem cells

Chromatin accessibility has been used to define how cells adopt region-specific neural fates. BAF45D is one of the subunits of a specialised chromatin remodelling BAF complex. It has been reported that BAF45D is expressed in spinal cord neural stem cells (NSCs) and regulates their fate specification. Within the developing vertebrate spinal cord, HOX genes exhibit spatially restricted expression patterns. However, the chromatin accessibility of BAF45D binding HOX genes in spinal cord NSCs is unclear. In the present study, we found that in H9-derived spinal cord NSCs, BAF45D targets TBX6, a gene that regulates spinal cord neural mesodermal progenitors. Furthermore, BAF45D binding to the NES gene is much more enriched in H9-derived spinal cord NSCs chromatin compared to ESCs chromatin. In addition, BAF45D binding to anterior and trunk/central HOX genes, but not to lumbosacral HOX genes, was much more enriched in NSCs chromatin compared to ESCs chromatin. These results may shed new light on the role of BAF45D in regulating region-specific spinal cord NSCs by targeting HOX genes.

Efficacy and Safety of Chimeric Antigen Receptor T Cells Therapy Strategy with Dual Targeting of CD19 and CD70 to Treat Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Background: Chimeric antigen receptor (CAR) T cells therapy is highly effective in the treatment of B-cell lymphoma, providing alternative therapeutic options for patients who failed to respond to conventional treatment or relapse. Despite impressive progress, more than 30-50% of patients treated with CD19 CAR-T cells experienced progressive disease. Antigen escape is one of the common causes of relapse, especially CD19-negative relapse. Absent or decreased cell surface CD19 as a mechanism of resistance after CD19 CAR-T cells treatment. CD70 is a novel and promising therapeutic target due to the restricted expression pattern in normal tissues and over expression in some lymphoma tissues. Our center explored the efficacy and safety of CAR-T cells therapy with dual targeting of CD19 and CD70 in the treatment of r/r lymphoma. Patients and Methods: The study included 8 relapsed/refractory diffuse large B-cell lymphoma patients, all patients have exhausted all available treatment options with progressive or stable disease and life expectancy >2 months were enrolled in the study. All patients expressed strong positivity of CD19 and CD70, all of patient details can be found in the table (Fig.1). Autologous T cells were apheresis collected and transduced with an apoptosis-inducible, safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/CD27/CD3z-iCasp9. All patients received cyclophosphamide/fludarabine chemotherapy conditioning 1-2 days before infusions of CAR-T cells. The quality of apheresis cells, efficiencies of gene transfer and T cell proliferation, CAR-T infusion dose and blood CAR copies were quantitatively documented. Results: In the first months after CAR-T cells infusion, 75.0% (6/8) of patients achieved complete response (CR), while 12.5% (1/8) of patients evaluated as partial response (PR) and 12.5% (1/8) of patients as progressive disease (PD), overall response rate (ORR) is 87.5% (7/8). In the evaluation of safety, 37.5% (3/8) of patients experienced cytokine release syndrome (CRS), and 25% (2/8) of patients experienced grade 1 CRS and 12.5% (1/8) of patients experienced grade 2 CRS, no case of severe CRS defined as≥grade 3 and no immune effector cell-associated neurotoxicity syndrome (ICANS) occurred. After a median follow-up of 19.9 months, 50% (4/8) of patients maintained CR, while 37.5% (3/8) of patients were relapsed, with a duration of response (DOR) of 50% (4/8), median DFS was 10.5 months and median OS was not reached (Fig.2). Conclusions: In summary, our research data confirms that the efficacy and safety of CD19/CD70 dual targeted CAR-T cells infusion, continued follow-up will determine whether the CD19/CD70 CAR-T cells therapy can obtain long term overall survival in our study. How to further improve the efficacy and safety of dual target CAR-T is still worth exploring. It's needed to optimize multi-specific targeting by CAR-T cells to improve the efficacy both in B cell malignancies and other hematological malignancies.

Association of DDX5/p68 protein with the upstream erythroid enhancer element (EHS1) of the gene encoding the KLF1 transcription factor

EKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. Regulation of KLF1 is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the DDX5/p68 protein as an activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site upstream enhancer element (EHS1). We further show that this protein associates with DEK and CTCF. We postulate that the range of interactions of DDX5/p68 with these and other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1 and enables the formation of a proper chromatin configuration at the Klf1 locus. These individual interactions provide quantitative contributions that, in sum, establish the high-level activity of the Klf1 promoter and suggest they can be selectively manipulated for clinical benefit.

The long non-coding RNA transcript, LOC100130460 (CAND1.11) gene, encodes a novel protein highly expressed in cancer cells and tumor human testis tissues.

Testis-specific genes encoding for long non-coding RNA (lncRNA) have been detected in several cancers; many produce proteins with restricted or aberrant expression patterns in normal or cancer tissues. To characterize new lncRNA involved in normal and/or pathological differentiation of testicular cells. Using bioinformatics analysis, we found that lncRNA LOC100130460 (CAND1.11) is expressed in normal and tumor testis; its expression was assessed in several human cell lines by qRT-PCR. CAND1.11 protein, produced by a single nucleotide mutation, was studied by western blot and immunofluorescence analysis on normal, classic seminoma, and Leydig cell tumor testicular tissues. CAND1.11 gene is primate-specific; its expression was low in SH-SY5Y cells and increased when differentiated with retinoic acid treatment. CAND1.11 expression in PC3 cells was higher than in PNT2 cells. CAND1.11 protein is present in the human testis and overexpressed in testicular cancer tissues. This report is one of the few providing evidence that a lncRNA produces a protein expressed in normal human tissues and overexpressed in several testicular cancers, suggesting its involvement in regulating cell proliferation and differentiation. Although further studies are needed to validate the results, our data indicate that CAND1.11 could be a potential new prognostic biomarker to use in proliferation and cancer.

Deciphering the impact of coding and non-coding SCN1A gene variants on RNA splicing.

Variants that disrupt normal pre-mRNA splicing are increasingly being recognized as a major cause of monogenic disorders. The SCN1A gene, a key epilepsy gene that is linked to various epilepsy phenotypes, is no exception. Approximately 10% of all reported variants in the SCN1A gene are designated as splicing variants, with many located outside of the canonical donor and acceptor splice sites, and most have not been functionally investigated. However, given its restricted expression pattern, functional analysis of splicing variants in the SCN1A gene could not be routinely performed. In this study, we conducted a comprehensive analysis of all reported SCN1A variants and their potential to impact SCN1A splicing and conclude that splicing variants are substantially misannotated and under-represented. We created a splicing reporter system consisting of 18 splicing vectors covering all 26 protein-coding exons with different genomic contexts and several promoters of varying strengths in order to reproduce the wild-type splicing pattern of the SCN1A gene, revealing cis-regulatory elements essential for proper recognition of SCN1A exons. Functional analysis of 95 SCN1A variants was carried out, including all 68 intronic variants reported in the literature, located outside of the splice sites canonical dinucleotides; 21 exonic variants of different classes (synonymous, missense, nonsense and in-frame deletion) and six variants observed in patients with epilepsy. Interestingly, almost 20% of tested intronic variants had no influence on SCN1A splicing, despite being reported as causative in the literature. Moreover, we confirmed that the majority of predicted exonic variants affect splicing unravelling their true molecular mechanism. We used functional data to perform genotype-phenotype correlation, revealing distinct distribution patterns for missense and splice-affecting 'missense' variants and observed no difference in the phenotype severity of variants leading to in-frame and out-of-frame isoforms, indicating that the Nav1.1 protein is highly intolerant to structural variations. Our work demonstrates the importance of functional analysis in proper variant annotation and provides a tool for high-throughput delineation of splice-affecting variants in SCN1A in a whole-gene manner.

Multimodal spatiotemporal transcriptomic resolution of embryonic palate osteogenesis

The terminal differentiation of osteoblasts and subsequent formation of bone marks an important phase in palate development that leads to the separation of the oral and nasal cavities. While the morphogenetic events preceding palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms driving the formation of this bony union of the fusing palate. Through bulk, single-nucleus, and spatially resolved RNA-sequencing analyses of the developing secondary palate, we identify a shift in transcriptional programming between embryonic days 14.5 and 15.5 pinpointing the onset of osteogenesis. We define spatially restricted expression patterns of key osteogenic marker genes that are differentially expressed between these developmental timepoints. Finally, we identify genes in the palate highly expressed by palate nasal epithelial cells, also enriched within palatal osteogenic mesenchymal cells. This investigation provides a relevant framework to advance palate-specific diagnostic and therapeutic biomarker discovery.

Coevolution of the Tlx homeobox gene with medusa development (Cnidaria: Medusozoa)

Cnidarians display a wide diversity of life cycles. Among the main cnidarian clades, only Medusozoa possesses a swimming life cycle stage called the medusa, alternating with a benthic polyp stage. The medusa stage was repeatedly lost during medusozoan evolution, notably in the most diverse medusozoan class, Hydrozoa. Here, we show that the presence of the homeobox gene Tlx in Cnidaria is correlated with the presence of the medusa stage, the gene having been lost in clades that ancestrally lack a medusa (anthozoans, endocnidozoans) and in medusozoans that secondarily lost the medusa stage. Our characterization of Tlx expression indicate an upregulation of Tlx during medusa development in three distantly related medusozoans, and spatially restricted expression patterns in developing medusae in two distantly related species, the hydrozoan Podocoryna carnea and the scyphozoan Pelagia noctiluca. These results suggest that Tlx plays a key role in medusa development and that the loss of this gene is likely linked to the repeated loss of the medusa life cycle stage in the evolution of Hydrozoa.

Juan Modolell (1937-2023).

Juan Modolell (1937-2023).

Genome-Wide Identification and Evolution Analysis of the CYP76 Subfamily in Rice (Oryza sativa).

The CYP76 subfamily, a member of the CYP superfamily, plays crucial roles in the biosynthesis of phytohormones in plants, involving biosynthesis of secondary metabolites, hormone signaling, and response to environmental stresses. Here, we conducted a genome-wide analysis of the CYP76 subfamily in seven AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, and Oryza glumaepatula. These were identified and classified into three groups, and it was found that Group 1 contained the largest number of members. Analysis of cis-acting elements revealed a large number of elements related to jasmonic acid and light response. The gene duplication analysis revealed that the CYP76 subfamily expanded mainly in SD/WGD and tandem forms and underwent strong purifying selection during evolution. Expression pattern analysis of OsCYP76s in various developmental stages revealed that the majority of OsCYP76s exhibit relatively restricted expression patterns in leaves and roots. We further analyzed the expression of CYP76s in O. sativa, japonica, and O. sativa, indica under cold, flooding, drought, and salt abiotic stresses by qRT-PCR. We found that OsCYP76-11 showed a huge increase in relative expression after drought and salt stresses. After flooding stress, OsiCYP76-4 showed a greater increase in expression compared to other genes. CYP76 in japonica and indica showed different response patterns to the same abiotic stresses, revealing functional divergence in the gene family during evolution; these may be the key genes responsible for the differences in tolerance to indica japonica. Our results provide valuable insights into the functional diversity and evolutionary history of the CYP76 subfamily and pave the way for the development of new strategies for improving stress tolerance and agronomic traits in rice.

Enhanced protein synthesis is a defining requirement for neonatal B cell development.

The LIN28B RNA binding protein exhibits an ontogenically restricted expression pattern and is a key molecular regulator of fetal and neonatal B lymphopoiesis. It enhances the positive selection of CD5+ immature B cells early in life through amplifying the CD19/PI3K/c-MYC pathway and is sufficient to reinitiate self-reactive B-1a cell output when ectopically expressed in the adult. In this study, interactome analysis in primary B cell precursors showed direct binding by LIN28B to numerous ribosomal protein transcripts, consistent with a regulatory role in cellular protein synthesis. Induction of LIN28B expression in the adult setting is sufficient to promote enhanced protein synthesis during the small Pre-B and immature B cell stages, but not during the Pro-B cell stage. This stage dependent effect was dictated by IL-7 mediated signaling, which masked the impact of LIN28B through an overpowering stimulation on the c-MYC/protein synthesis axis in Pro-B cells. Importantly, elevated protein synthesis was a distinguishing feature between neonatal and adult B cell development that was critically supported by endogenous Lin28b expression early in life. Finally, we used a ribosomal hypomorphic mouse model to demonstrate that subdued protein synthesis is specifically detrimental for neonatal B lymphopoiesis and the output of B-1a cells, without affecting B cell development in the adult. Taken together, we identify elevated protein synthesis as a defining requirement for early-life B cell development that critically depends on Lin28b. Our findings offer new mechanistic insights into the layered formation of the complex adult B cell repertoire.

The RNA helicase DDX3 and its role in c-MYC driven germinal center-derived B-cell lymphoma.

DDX3X is an RNA helicase with many functions in RNA metabolism such as mRNA translation, alternative pre-mRNA splicing and mRNA stability, but also plays a role as a regulator of transcription as well as in the Wnt/beta-catenin- and Nf-κB signaling pathways. The gene encoding DDX3X is located on the X-chromosome, but escapes X-inactivation. Hence females have two active copies and males only one. However, the Y chromosome contains the gene for the male DDX3 homologue, called DDX3Y, which has a very high sequence similarity and functional redundancy with DDX3X, but shows a more restricted protein expression pattern than DDX3X. High throughput sequencing of germinal center (GC)-derived B-cell malignancies such as Burkitt Lymphoma (BL) and Diffuse large B-cell lymphoma (DLBCL) samples showed a high frequency of loss-of-function (LOF) mutations in the DDX3X gene revealing several features that distinguish this gene from others. First, DDX3X mutations occur with high frequency particularly in those GC-derived B-cell lymphomas that also show translocations of the c-MYC proto-oncogene, which occurs in almost all BL and a subset of DLBCL. Second, DDX3X LOF mutations occur almost exclusively in males and is very rarely found in females. Third, mutations in the male homologue DDX3Y have never been found in any type of malignancy. Studies with human primary GC B cells from male donors showed that a loss of DDX3X function helps the initial process of B-cell lymphomagenesis by buffering the proteotoxic stress induced by c-MYC activation. However, full lymphomagenesis requires DDX3 activity since an upregulation of DDX3Y expression is invariably found in GC derived B-cell lymphoma with DDX3X LOF mutation. Other studies with male transgenic mice that lack Ddx3x, but constitutively express activated c-Myc transgenes in B cells and are therefore prone to develop B-cell malignancies, also showed upregulation of the DDX3Y protein expression during the process of lymphomagenesis. Since DDX3Y is not expressed in normal human cells, these data suggest that DDX3Y may represent a new cancer cell specific target to develop adjuvant therapies for male patients with BL and DLBCL and LOF mutations in the DDX3X gene.

Antigene MYCN Silencing by BGA002 Inhibits SCLC Progression Blocking mTOR Pathway and Overcomes Multidrug Resistance.

Small-cell lung cancer (SCLC) is the most aggressive lung cancer type, and is associated with smoking, low survival rate due to high vascularization, metastasis and drug resistance. Alterations in MYC family members are biomarkers of poor prognosis for a large number of SCLC. In particular, MYCN alterations define SCLC cases with immunotherapy failure. MYCN has a highly restricted pattern of expression in normal cells and is an ideal target for cancer therapy but is undruggable by traditional approaches. We propose an innovative approach to MYCN inhibition by an MYCN-specific antigene-PNA oligonucleotide (BGA002)-as a new precision medicine for MYCN-related SCLC. We found that BGA002 profoundly and specifically inhibited MYCN expression in SCLC cells, leading to cell-growth inhibition and apoptosis, while also overcoming multidrug resistance. These effects are driven by mTOR pathway block in concomitance with autophagy reactivation, thus avoiding the side effects of targeting mTOR in healthy cells. Moreover, we identified an MYCN-related SCLC gene signature comprehending CNTFR, DLX5 and TNFAIP3, that was reverted by BGA002. Finally, systemic treatment with BGA002 significantly increased survival in MYCN-amplified SCLC mouse models, including in a multidrug-resistant model in which tumor vascularization was also eliminated. These findings warrant the clinical testing of BGA002 in MYCN-related SCLC.

Abstract B022: The adaptor protein FRS2beta fashions a cytokine-rich inflammatory microenvironment that is associated with DCIS and promotes breast cancer carcinogenesis

Abstract Although it is commonly held that pro-inflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we report that FRS2beta, an adaptor protein with a highly restricted epithelial expression pattern, mediates the pro-inflammatory changes that alter the stroma in premalignant mammary tissues. We posit that these changes, which include lesions of ductal carcinoma in situ (DCIS), precede disease onset. In the MMTV-ErbB2 mouse model of mammary carcinoma, FRS2beta-deficiency markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2beta-deficient premalignant tissues. We found that the co-localization of FRS2beta and the NEMO subunit of the IkappaB kinase (IKK) complex in early endosomes activated nuclear factor-kappaB (NFkappaB), a master regulator of inflammation. Moreover, the inhibition of NFkappaB-induced cytokines CXC chemokine ligand (CXCL)12 and insulin-like growth factor (IGF1) abrogated tumorigenesis. In line with this, human breast cancer tissues that express higher levels of FRS2beta contain more stroma. The elucidation of the FRS2beta-NFkappaB axis uncovers a hitherto unknown molecular link between the pro-inflammatory changes in the DCIS and the disease onset. Citation Format: Noriko Gotoh. The adaptor protein FRS2beta fashions a cytokine-rich inflammatory microenvironment that is associated with DCIS and promotes breast cancer carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B022.

Risk Factors, Hyaluronidase Expression, and Clinical Immunogenicity of Recombinant Human Hyaluronidase PH20, an Enzyme Enabling Subcutaneous Drug Administration

Multiple FDA-approved and clinical-development stage therapeutics include recombinant human hyaluronidase PH20 (rHuPH20) to facilitate subcutaneous administration. As rHuPH20-reactive antibodies potentially interact with endogenous PH20, we investigated rHuPH20 immunogenicity risk through hyaluronidase tissue expression, predicted B cell epitopes, CD4+ T cell stimulation indices and related these to observed clinical immunogenicity profiles from 18 clinical studies. Endogenous hyaluronidase PH20 expression in humans/mice was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and deep RNA-Seq. rHuPH20 potential T cell epitopes were evaluated in silico and confirmed in vitro. Potential B cell epitopes were predicted for rHuPH20 sequence in silico, and binding of polyclonal antibodies from various species tested on a rHuPH20 peptide microarray. Clinical immunogenicity data were collected from 2643 subjects. From 57 human adult and fetal tissues previously screened by RT-PCR, 22 tissue types were analyzed by deep RNA-Seq. Hyaluronidase PH20 messenger RNA expression was detected in adult human testes. In silico analyses of the rHuPH20 sequence revealed nine T cell epitope clusters with immunogenic potential, one cluster was homologous to human leukocyte antigen. rHuPH20 induced T cell activation in 6–10% of peripheral blood mononuclear cell donors. Fifteen epitopes in the rHuPH20 sequence had the potential to cross-react with B cells. The cumulative treatment-induced incidence of anti-rHuPH20 antibodies across clinical studies was 8.8%. Hyaluronidase PH20 expression occurs primarily in adult testes. Low CD4+ T cell activation and B cell cross-reactivity by rHuPH20 suggest weak rHuPH20 immunogenicity potential. Restricted expression patterns of endogenous PH20 indicate low immunogenicity risk of subcutaneous rHuPH20.Graphical abstract

Temporal analysis of enhancers during mouse cerebellar development reveals dynamic and novel regulatory functions.

We have identified active enhancers in the mouse cerebellum at embryonic and postnatal stages which provides a view of novel enhancers active during cerebellar development. The majority of cerebellar enhancers have dynamic activity between embryonic and postnatal development. Cerebellar enhancers were enriched for neural transcription factor binding sites with temporally specific expression. Putative gene targets displayed spatially restricted expression patterns, indicating cell-type specific expression regulation. Functional analysis of target genes indicated that enhancers regulate processes spanning several developmental epochs such as specification, differentiation and maturation. We use these analyses to discover one novel regulator and one novel marker of cerebellar development: Bhlhe22 and Pax3, respectively. We identified an enrichment of de novo mutations and variants associated with autism spectrum disorder in cerebellar enhancers. Furthermore, by comparing our data with relevant brain development ENCODE histone profiles and cerebellar single-cell datasets we have been able to generalize and expand on the presented analyses, respectively. We have made the results of our analyses available online in the Developing Mouse Cerebellum Enhancer Atlas, where our dataset can be efficiently queried, curated and exported by the scientific community to facilitate future research efforts. Our study provides a valuable resource for studying the dynamics of gene expression regulation by enhancers in the developing cerebellum and delivers a rich dataset of novel gene-enhancer associations providing a basis for future in-depth studies in the cerebellum.