Journal: bioRxiv

Article Title: Transcription Factor Subtype Governs Response and Resistance to DLL3-Directed T-Cell Engagement in Small Cell Lung Cancer

doi: 10.64898/2026.04.02.715020

Figure Lengend Snippet: (A) , (I) , (M) Swimmer plot showing the clinical course, treatment outcomes, and tumor fraction for patients 6527 ( A ), 5685 ( I ), and 7373 ( M ). ( B ) Scatterplot showing the Z-score of gene expression at C1D1 vs. at C9D1 for patient 6527. Each point represents a single gene. Genes (black) represent key genes of interest in SCLC. Genes classified as higher in C1D1 (dark blue) show a ≥2-unit increase in Z-score at C1D1 compared to C9D1, whereas genes higher in C9D1 (dark green) show a ≥2-unit increase in Z-score at C9D1 compared to C1D1. Other genes (grey) do not exhibit a substantial change between timepoints. The dashed diagonal line represents no change between timepoints. ( C ) Genome browser view of H3K4me3 cfChIP-seq signal at C1D1 (dark blue) and at C9D1 (dark green). ( D ) Line plot showing the dynamics of SCLC-N and SCLC-A transcriptional signatures ( Methods ) over serial timepoints for patient 6527. The thick colored lines represent the median expression per timepoint. Shaded ribbons indicate the interquartile range (25th–75th percentile). At each timepoint, the statistical difference between SCLC-N and SCLC-A signatures was assessed using a two-sided Wilcoxon rank-sum test, with significance indicated above the timepoints: * (p < 0.05), ** (p < 0.01), **** (p < 0.0001), **** (p < 0.0001); ns = not significant. SCLC-N is shown in dark green and SCLC-A in dark blue. ( E ) Scatter plot showing gene-level Z-scores at baseline vs progression for patient 6527. Each point represents one gene. Genes annotated as SCLC-A or SCLC-N according to predefined subtype gene lists are highlighted (SCLC-A, dark blue; SCLC-N, dark green), while all other genes are shown in grey. Subtype centroids (mean Z-score across genes within each subtype) are shown as crosses. The annotated ΔZ values indicate the mean change in Z-score (C9D1 − C1D1) for each subtype. ( F ) Heatmap showing the top 15 most variable MSigDB Hallmark pathways based on mean Z-scores across serial timepoints for patient 6527. Pathways are sorted by the absolute difference between C1D1 and C9D1. ( G ), Genome-wide copy number variation profiles for patient 6527 at C1D1 and at C9D1. ( H ) Hypothetical clonal evolution (“fish plot”) for patient 6527 across longitudinal sampling. Colored areas represent the estimated fraction of each clone at each timepoint (light blue, clone 1/ancestral; dark blue, clone 2; green, clone 3; light green, clone 4). Clonal relationships are depicted by nesting according to the specified parent structure (clone 1 is the founder clone; clones 2 and 3 arise from clone 1; clone 4 arises from clone 3). ( J ) Scatterplot showing the Z-score of gene expression at C1D1 vs. at C14D15 for patient 5685. Each point represents a single gene. Genes (black) represent key genes of interest in SCLC. Genes classified as higher in C1D1 (light blue) show a ≥2-unit increase in Z-score at C1D1 compared to C14D15, whereas genes higher in C14D15 (orange) show a ≥2-unit increase in Z-score at C14D15 compared to C1D1. Other genes (grey) do not exhibit a substantial change between timepoints. The dashed diagonal line represents no change between timepoints. ( K ) Genome browser view of H3K4me3 cfChIP-seq signal at C1D1 (light blue) and at C14D15 (orange). ( L ) Representative IHC for ASCL1, NEUROD1, POU2F3, and DLL3 from patient 5685 on C14D15 showing loss of DLL3 protein expression. ( N ) Genome browser view of cfChIP–seq H3K4me3 signal across representative loci for patient 7373. Of note, there was no baseline plasma sample available for this patient. ( O ) scRNAseq UMAP visualization of all CD3+ T cells (n=1040 CD3+ T cells) from adrenalectomy. NKT: Natural Killer T-cells, NK: Natural Killer, T reg: Regulatory T cells. ( P ) scRNAseq UMAP visualization of all CD3+ T cells showing expression of regulatory T cell markers and markers of T cell exhaustion: FOXP3, CTLA4, TIGIT, and LAG3. ( Q ) Representative multiplex immunofluorescence (mIF) staining of the adrenalectomy specimen of patient 7373.

Article Snippet: Primers; Ascl1 (Mm0358063_m1), Neurod1 (Mm01280117_m1), Dll3 (Mm00432854_m1), ActinB (Mm00607939_s1).

Techniques: Gene Expression, Expressing, Genome Wide, Sampling, Clone Assay, Clinical Proteomics, Multiplex Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Transcription Factor Subtype Governs Response and Resistance to DLL3-Directed T-Cell Engagement in Small Cell Lung Cancer

doi: 10.64898/2026.04.02.715020

Figure Lengend Snippet: ( A ) An acquired Tarlatamab-resistant tumor (1014P2-TR#2) was harvested for downstream tumor analyses, and a stable cell line was established for subsequent experiments. ( B ) Representative flow cytometric histogram for cell-surface DLL3 expression in the parental 1014P2 and 1014P2-TR cell line from three independant experiments. For (b), y-axis is normalized to mode. ( C ) Immunoblot analysis of parental 1014P2 cells and 1014P2-TR#2 cells with the antibodies indicated. ( D ) IHC staining for ASCL1, NEUROD1, and DLL3 in a vehicle-treated 1014P2 tumor (left) and the 1014P2-TR#2 tumor (right). Black box on top row figures indicate the area magnified on the bottom row. Original magnification 5x for top row and 40x for bottom row. ( E ) Heatmap of top differentially expressed genes (p value<0.05) from RNA-seq comparing 1014P2-TR#2 cells (2 biological replicates) vs. parental 1014P2 cells (2 biological replicates) (total 714 genes). Neurod1 and EMT genes are labeled. ( F ) Hallmarks gene set enrichment analysis of RNA-seq data from of top enriched gene sets in 1014P2-TR#2 cells vs. parental 1014P2 cells. NES = Normalized Enrichment Score. ( G ) Gene set enrichment plot of ASCL1 (left) and NEUROD1 (right) gene signatures (see Method ) using H3K27ac ChIP-seq data comparing 1014P2-TR#2 vs. 1014P2. ( H ) In vivo tarlatamab competition experiments where 1014P2 and 1014P2-TR#2 cells were subcutaneously injected into opposite bilateral flanks of the same humanized CD3 BL6 mouse (one cell line per flank; n=2 independent mice, one genOway and one Biocytogen mouse) and treated with tarlatamab (0.3 mg/kg) by IP injection on days 0, 7, and 14 (green arrow head). ( I ) Tumors from were harvested at experimental endpoint (>2000 mm 3 ) and analyzed by flow cytometry for GFP (for tumor cells), and CD45+, CD3+, CD4+, CD8+ for immune cell infiltration. ( J ) IHC for CD3 and CD8 of 1014P2 parental tumor and 1014P2-TR#2 tumor from experiment in . Black box indicates the area magnified on bottom right corner of each image. Black bar is 100 mm for weak magnification (original maginification 5x) and 25 mm for magnified image (original magnification 40x). ( K ) Parental 1014P2 and 1014P2-TR#2 cells were injected subcutaneously into individual humanized CD3 mice and treated with tarlatamab (0.3 mg/kg) administered weekly on days 0, 7, 14, and 21 (green arrow head). Left panel shows individual tumor growth and right panel shows mean +/- SEM. Two-way ANOVA followed by Sidak’s multiple-comparisons test was conducted ((**p < 0.01, n=16 tumors (eight mice) for parental 1014P2 and n=8 tumors (four mice) for 1014P2-TR#2)).( L ) Immunoblot analysis for ASCL1, NEUROD1, and DLL3 from 1014P2 and 1014P2 tarlatamab resistant cell lines (TR#2, 4R, 4L, 5, 5H, and 6) established from tarlatamab resistant tumors. Details of each resistant tumor cell line are in the methods section. ( M ) Tumors from 1014P2 resistant tumors (TR#2, 5, 5H, and 6) were harvested and dissociated into single cell to assess FOXP3 expression on CD4+ T cells (left panel) and PD1/TIM3/LAG3 expression on CD8+ T cells (right panel). ( N ), Immunoblot analysis of parental 1014P2 cells, 1014P2 cells transduced with NEUROD1 overexpression (OE) vector, and mouse tumors made from 1014P2 NEUROD1 overexpression cell line treated with either vehicle or tarlatamab. Of note, the Figure S5H immunoblot contains the same 1014P2 NEUROD1 overexpression mouse tumors with additional controls. ( O ) Schematic of the proposed model in which ASCL1-driven, DLL3-high SCLCs are highly sensitive to tarlatamab. This response requires tumor cell expression of DLL3 driven by ASCL1 and functional CD3⁺ T cells. Tarlatamab exerts both tumor-intrinsic and tumor-extrinsic selective pressure to overcome its mechanism of action. Tumor cells undergo subtype selection toward NEUROD1, which promotes loss of DLL3 and can ultimately lead to acquired resistance. In parallel, tarlatamab promotes enrichment of regulatory and exhausted T cells, compromising CD3 effector function and thereby limiting therapeutic efficacy. Although both mechanisms can be selected under therapeutic pressure, one mechanism of acquired resistance ultimately predominates within a given tumor to sustain tumor growth in the presence of tarlatamab. Figure made using BioRender.

Article Snippet: Primers; Ascl1 (Mm0358063_m1), Neurod1 (Mm01280117_m1), Dll3 (Mm00432854_m1), ActinB (Mm00607939_s1).

Techniques: Stable Transfection, Expressing, Western Blot, Immunohistochemistry, RNA Sequencing, Labeling, ChIP-sequencing, In Vivo, Injection, Flow Cytometry, Single Cell, Transduction, Over Expression, Plasmid Preparation, Functional Assay, Selection, Drug discovery

Journal: Frontiers in Transplantation

Article Title: Mechanisms inducing differentiation of adult islet progenitor-like cells into functional islet-like organoids

doi: 10.3389/frtra.2026.1740314

Figure Lengend Snippet: Requirement of CN/NFAT signaling to recruit p300 to RFX6 and NEUROD1 promoters during ISX9-induced IPC islet organoid differentiation (A) induction of NFAT family isoform genes in IPC clusters treated with ISX9 for 24 h in the presence of CN inhibitor FK506. (B) RFX6 and NEUROD1 promoter activation by ISX9 in IPCs in the presence of FK506 or transfected with gene vectors overexpressing dominant negative NFAT (dnNFAT) and mutated control (dnNFATm). (C) ChIP assay of association of NFATC2, p300, HDAC1, HDAC2, and HDAC3 with RFX6, NEUROD1, and NEUROG3 promoters upon 6 h treatment of IPCs with ISX9 with and without 24 h pretreatment with ITF2357. Graphed values are expressed as mean ± SD. Asterisks above bars indicate statistically significant differences (* p < 0.05, *** p < 0.001) in mean values for treatments based on a two-way ANOVA and Sidak's multiple comparison test. Data shown are results from at least three independent experiments using IPCs derived from three individual donors.

Article Snippet: Gluc-ON reporters for RFX6 (HPRM53326-PG04), NEUROD1 (HPRM69533-PG04), and INS (HPRM30189-PG04) promoters were obtained from GeneCopoeia.

Techniques: Activation Assay, Transfection, Dominant Negative Mutation, Control, Comparison, Derivative Assay

Journal: Frontiers in Transplantation

Article Title: Mechanisms inducing differentiation of adult islet progenitor-like cells into functional islet-like organoids

doi: 10.3389/frtra.2026.1740314

Figure Lengend Snippet: Schematic overview of IPC isolation, expansion, and differentiation. Adult human pancreatic tissue obtained from islet cell isolation fractions was expanded in vitro to generate a CD9 + PROCR + IPC-enriched population. IPCs were identified using transcriptomic and phenotypic analyses (scRNA-seq, flow cytometry, and immunofluorescence). An RGS16 + organoid-forming subset was characterized within the expanded population. IPC clusters were subsequently subjected to ISX9-mediated differentiation. ISX9 treatment stimulated calcineurin (CN)/NFAT signaling, promoted NFATC2–p300 association and binding at RFX6 and NEUROD1 promoters, and induced downstream endocrine transcriptional programs (NGN3, RFX6, NEUROD1, NKX2.2, NKX6.1, MAFA), resulting in functional islet organoids.

Article Snippet: Gluc-ON reporters for RFX6 (HPRM53326-PG04), NEUROD1 (HPRM69533-PG04), and INS (HPRM30189-PG04) promoters were obtained from GeneCopoeia.

Techniques: Isolation, Cell Isolation, In Vitro, Flow Cytometry, Immunofluorescence, Binding Assay, Functional Assay

Journal: eLife

Article Title: Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels

doi: 10.7554/eLife.108485

Figure Lengend Snippet: ( A ) Transcriptional start site enrichment profiles of scATAC-Seq datasets. Lines are colored by time points. ( B ) Fragment size distribution of scATAC-Seq datasets. Individual time points are indicated by colored lines. ( C ) Heatmap showing the Pearson correlation between gene expression and gene accessibility for each retina cell type. ( D ) Heatmap of cell type-specific genes. ( E ) Heatmap of cell type-specific peaks. ( F ) Heatmap of cell type-specific motifs. ( G ) Examples of transcription factor (TF) footprint profiles for Pou4f2, Crx, Nfix, and Onecut1 in indicated scATAC-Seq cell types. ( H ) Examples of chromVAR score are shown for Otx2, Pou2f2, Nfix, and Neurod1 using scATAC-Seq datasets. ( I ) The relative abundance of retinal cell types in scRNA- and scATAC-Seq is different between developing 13LGS and mouse retina.

Article Snippet: Antibody , NeuroD1 (E3E4F) Rabbit mAb , Cell Signaling , 62953 , 0.5 μg/reaction.

Techniques: Gene Expression

Journal: eLife

Article Title: Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels

doi: 10.7554/eLife.108485

Figure Lengend Snippet: ( A ) Schematic illustrating annotation of cis -regulatory elements in RPCs and photoreceptor precursors by integration of scATAC-Seq and CUT&RUN 13LGS and mouse datasets. ( B ) Heatmaps show annotated accessible regulatory elements in both 13LGS and mouse. Promoters, activated enhancers (AEs), and poised enhancers (PEs), which are associated with histone markers associated with genes in clusters C2 and C3, which are selectively active in 13LGS RPCs and/or photoreceptor precursors. Shading indicates CUT&TAG signal for the corresponding histone modification within 2 kb of the scATAC-Seq peak center. Bar plots displaying the number of each category of regulatory element in each species that are conserved or species-specific. ( C ) Dot plots showing the enrichment of binding sites for Otx2 and Neurod1, TFs which are broadly expressed in both neurogenic RPC and photoreceptor precursors, which are enriched in both conserved cis -regulatory elements in both species. ( D ) Bar plots showing the number of conserved and species-specific enhancers per transcription start site (TSS) in four cone-promoting genes between 13LGS and mouse. ( E ) The gene regulatory networks (GRNs) regulating Thrb expression in 13LGS and mouse late N. RPCs. ( F ) An example of a Thrb-related regulon and its corresponding scATAC-Seq and CUT&RUN tracks. The arrow indicates the consistent regulatory relationships between GRN prediction and experimental validations. ( G ) The epigenetic model of cone specification in 13LGS and mouse.

Article Snippet: Antibody , NeuroD1 (E3E4F) Rabbit mAb , Cell Signaling , 62953 , 0.5 μg/reaction.

Techniques: Modification, Binding Assay, Expressing

Journal: eLife

Article Title: Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels

doi: 10.7554/eLife.108485

Figure Lengend Snippet: ( A ) Transcriptional start site enrichment profiles of scATAC-Seq datasets. Lines are colored by time points. ( B ) Fragment size distribution of scATAC-Seq datasets. Individual time points are indicated by colored lines. ( C ) Heatmap showing the Pearson correlation between gene expression and gene accessibility for each retina cell type. ( D ) Heatmap of cell type-specific genes. ( E ) Heatmap of cell type-specific peaks. ( F ) Heatmap of cell type-specific motifs. ( G ) Examples of transcription factor (TF) footprint profiles for Pou4f2, Crx, Nfix, and Onecut1 in indicated scATAC-Seq cell types. ( H ) Examples of chromVAR score are shown for Otx2, Pou2f2, Nfix, and Neurod1 using scATAC-Seq datasets. ( I ) The relative abundance of retinal cell types in scRNA- and scATAC-Seq is different between developing 13LGS and mouse retina.

Article Snippet: Then 0.5 μg of the following antibodies were added to each respective reaction: IgG control (EpiCypher, 13-0042), H3K4me1 (EpiCypher, 13-0057), H3K4me3 (EpiCypher, 13-0041), H3K27ac (EpiCypher, 13-0059), H3K27me3 (EpiCypher, 13-0055), NeuroD1 (Cell Signaling, 62953), Otx2 (R&D Systems, BAF1979), or Otx2 (Atlas Antibodies, HPA000633).

Techniques: Gene Expression

Journal: eLife

Article Title: Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels

doi: 10.7554/eLife.108485

Figure Lengend Snippet: ( A ) Schematic illustrating annotation of cis -regulatory elements in RPCs and photoreceptor precursors by integration of scATAC-Seq and CUT&RUN 13LGS and mouse datasets. ( B ) Heatmaps show annotated accessible regulatory elements in both 13LGS and mouse. Promoters, activated enhancers (AEs), and poised enhancers (PEs), which are associated with histone markers associated with genes in clusters C2 and C3, which are selectively active in 13LGS RPCs and/or photoreceptor precursors. Shading indicates CUT&TAG signal for the corresponding histone modification within 2 kb of the scATAC-Seq peak center. Bar plots displaying the number of each category of regulatory element in each species that are conserved or species-specific. ( C ) Dot plots showing the enrichment of binding sites for Otx2 and Neurod1, TFs which are broadly expressed in both neurogenic RPC and photoreceptor precursors, which are enriched in both conserved cis -regulatory elements in both species. ( D ) Bar plots showing the number of conserved and species-specific enhancers per transcription start site (TSS) in four cone-promoting genes between 13LGS and mouse. ( E ) The gene regulatory networks (GRNs) regulating Thrb expression in 13LGS and mouse late N. RPCs. ( F ) An example of a Thrb-related regulon and its corresponding scATAC-Seq and CUT&RUN tracks. The arrow indicates the consistent regulatory relationships between GRN prediction and experimental validations. ( G ) The epigenetic model of cone specification in 13LGS and mouse.

Article Snippet: Then 0.5 μg of the following antibodies were added to each respective reaction: IgG control (EpiCypher, 13-0042), H3K4me1 (EpiCypher, 13-0057), H3K4me3 (EpiCypher, 13-0041), H3K27ac (EpiCypher, 13-0059), H3K27me3 (EpiCypher, 13-0055), NeuroD1 (Cell Signaling, 62953), Otx2 (R&D Systems, BAF1979), or Otx2 (Atlas Antibodies, HPA000633).

Techniques: Modification, Binding Assay, Expressing