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PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
In vivo BLI was performed weekly until mice developed endpoint criteria (i.e., body condition scoring <2 or significantly high bioluminescence (BL) signals on the whole body of >1010 radiance).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
We made the region of interest (ROI) in BLI to cover the whole body to measure the total flux (photons/sec, p/s).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Mice with no detectable signal by the fourth-week post-injection were scored ‘ungrafted’.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
All other mice were monitored for tumor growth and survival.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
BL signals in U2932-Luc cells were observed from the second-week post-injection in both NSG-IL6 and NSG-IL6/SGM3 mice (Figure 1A,B).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
As the DLBCL growth progressed, the BL signals increased, and the focal BL signal spread throughout the body.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
While the BL of VAL-Luc cells was also observed from the second-week post-injection in NSG and NSG-IL6 mice, rapid tumor progression was noticed in comparison to the U2932-Luc cells.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Most mice (9/11 mice) exhibited a high tumor burden by 4th week (Figure 1C,D).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Of note, in further contrast to the U2932-Luc cells, there was no detectable engraftment of VAL-Luc cells in NSG-IL6/SGM3 mice despite some proliferation of cells near the injection site in weeks 6 and 7 (Figure 1C,D).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
These data suggest that the NSG-IL6 mice are highly permissive to both ABC and GCB subtypes of DLBCL cell lines.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Furthermore, no engraftment for either U2932 or VAL cells was found in the NSG-SGM3 mouse strain (Figure 1A,C).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
In U2932-Luc cell-injected NSG mice, BL signals were shown in some mice (15/30 mice), whereas tumor growth in VAL-Luc-injected NSG mice rapidly progressed, and all the mice died by the 3rd week (Figure 1A,C).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
The rapid disease progression of VAL-Luc NSG-IL6-xenografted mice relative to the U2932-Luc NSG-IL6-xenografted mice corresponded to a poorer survival (Figure 2A; p = 0.0001; median survival 24 days vs. 49 days).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
We also observed a 75% (15/20) engraftment rate for U2932-Luc cells in NSG-IL6 mice and 32% (6/19) in NSG-IL6/SGM3 mice compared to 82% (9/11) for VAL-Luc cell engraftment in NSG-IL6 mice and 0% (0/10) in NSG-IL6/SGM3 mice (Figure 2B,C).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Of note, both DLBCL cell lines in NSG-IL6 mice demonstrated uniform progression to death in the late phase of the disease.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
All mice injected with U2932-Luc and VAL-Luc died in a 16- and 9-day window, respectively.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Previously, the subcutaneous passaged WSU in NOD/-scid mice demonstrated lymph node infiltration .
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
U2932 and primary DLBCL cells in the MISTRG mice engrafted with cord blood human hematopoietic stem (HSC) cells or non-engrafted MISTRG6 showed spleen infiltration .
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
To assess organ-specific engraftment of DLBCL cells in NSG-IL6 mice, we euthanized four xenografted mice per week at weeks 2–6 post-injection and performed ex vivo imaging of the excised organs.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
U2932-Luc cells were initially found in the spleen, liver, and lungs starting in week 2 (Figure 3A,B, left panels).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Subsequently, U2932 cells were found in the ovaries of female mice, which is reflective of the parental tumor , spine, and brain (Supplementary Figure S1).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
VAL-Luc cells also showed a similar pattern but occurred earlier (Figure 3D,E, right panels).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Both U2932-Luc and VAL-Luc cells in NSG-IL6 mice exhibited a highly uniform tissue tropism that was unique to each cell line (spleen > liver > lung > brain > ovary).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Next, we confirmed U2932-Luc and VAL-Luc infiltrations into the initially targeted organs and spleen, liver, and lung tissues by immunohistochemistry (IHC) staining for the human CD20 B cell surface marker.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
IHC staining showed a low CD20 positive cell infiltrate at the second-week post-injection that steadily increased at the third- and fourth-week post-injection (Figure 3C,F), consistent with the BL imaging (Figure 1 and Figure 3A,D).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Notably, the NSG-IL6 mouse spleens at week 4 were dominated by the U2932-Luc cells.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
These findings confirm the early and highly permissive IL6-expressing mice for the engraftment and growth of DLBCL cells in organs consistent with clinical disease progression.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
To confirm evidence that IL6 signaling is a critical driver of a subset of DLBCL and an indicator of engraftment and growth in NSG-IL6 mice , we treated U2932 and VAL cells with human IL6 and determined STAT3 activation.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
We observed STAT3 activation in U2932 cells after 1 h incubation with human IL6, in agreement with the previous report, and further showed that phosphorylation of STAT3 (pSTAT3) is inducible at a higher abundance after 30 min (Figure 4A).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Although VAL cells show the constitutive activation of STAT3 as indicated by the presence of pSTAT3 in untreated cells, IL6 promotes additional STAT3 activation by ~6-fold after 30 min and to a lesser extent after 1 h. Previous work linked the expression of the IL6 receptor (IL6R) α subunit (IL6Rα) and gp130, the signaling chain subunit of the IL6R, to IL6-mediated STAT3 activation in DLBCL .
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
We confirmed that both IL6Rα and gp130 are expressed in U2932 and VAL cells, and we interestingly found VAL cells to express a significantly higher abundance of both subunits (Supplementary Figure S2).
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
While human IL6 did not significantly increase U2932 cell proliferation, human IL6 increased VAL cell proliferation (Figure 4B), supporting the enhanced growth of VAL-Luc cells engrafted in NSG-IL6 mice compared to U2932-Luc cells.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
However, the basal proliferation of VAL cells compared to U2932 cells was significantly higher (p = 0.01, area under the curve), most likely owing to the aggressive proliferative phenotype associated with the so-called double- or triple-hit (BCL2/BCL6/MYC translocation, ) positive VAL cells, and thus, it also contributes to the more rapid expansion in NSG-IL6 mice.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
We developed a humanized mouse model that supports DLBCL engraftment and expansion in secondary lymphoid organs and extranodal sites (e.g., liver and lung) consistent with clinical presentation and further confirmed the pivotal role of human IL6 in human DLBCL cell homing in mice.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
A previous study demonstrated that ABC DLBCL cell lines engrafted and colonized the bone marrow and spleen only when MISTRG was engrafted with human HSCs or human IL6 was expressed in MISTRG (MISTRG6) .
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
These data suggest that human IL6 provides a more fertile environment for DLBCL cells, particularly subsets with inherent responsiveness to IL6 signaling, to engraft and expand in an in vivo model.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
However, it raises another question whether human IL6 alone is sufficient for the homing of human DLBCL cells in immune-deficient mice.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Here, we evaluated DLBCL colonization and expansion in NSG mice expressing human IL3, CSF2, and KITLG with and without human IL6 co-expression and also in IL6-only-expressing NSG mice.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Our in vivo studies show that human IL6-alone-expressing NSG mice are permissive to the engraftment and organ colonization of the U2932 ABC and VAL GCB DLBCL cells, strongly indicating that IL6 is sufficient for systemic DLBCL tumor growth.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Unlike MISTRG, a similar mouse, NSG-SGM3 mice, exhibited no engraftment of either ABC or GCB cells, whereas in the case of the NSG-alone engraftment of ABC cells was poor (50%), while GCB cell engraftment was rapid and vigorous.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Such discrepancy may be derived from differences of other human cytokines or mouse backgrounds.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
However, without engraftment success rates in MISTRG or MISTRG6, we could not explain such a difference.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Of interest, the NSG-IL6/SGM3 mice were somewhat permissive to U2932 engraftment (32%) and steadily increased tumor burden, leading to worsening survival.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Although traditional subcutaneous xenograft DLBCL mouse models allow for adequate drug discovery studies, the major limitation is the localized tumor growth in an often clinically non-relevant tissue that constrains the translation of how effectively new therapeutic strategies control tumor burden in humans.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
An in vivo model that more closely recapitulates the clinical progression of DLBCL within tissues that parallel the human disease provides a substantial advantage over these models.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
While spontaneous mouse lymphoma models overcome this limitation, these mouse tumors typically take months to develop and are driven by the enforced expression of specific oncogenes that fail to represent the molecular heterogeneity of DLBCL between subtypes and within a given tumor.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Our approach of an intravenous injection of Luciferase-expressing human DLBCL cells in human IL6 transgenic NSG mice demonstrated rapid tumor cell engraftment and expansion, showing unique organ infiltration patterns reflecting the DLBCL subtype.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
We observed DLBCL cell infiltration and growth as early as two weeks post-injection, significantly faster than the MISTRG6 model .
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
Furthermore, despite the previous survey of GCB DLBCL cell lines demonstrating this subtype displays little to no IL6R expression, we show the VAL GCB DLBCL cells express a high abundance of both IL6R subunits.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
This finding suggests that not all GCB DLBCL cells are deficient in IL6 signaling and perhaps supports yet another aggressive phenotype of the double- or triple-hit DLBCL subset.
PMC11394112
Development of New Diffuse Large B Cell Lymphoma Mouse Models
In summary, we establish an intravenous xenograft model for DLBCL that shortens the time to tumor development (median survival of U2932-Luc and VAL-Luc in NSG-IL6 mice; 49 days and 24 days), shows highly uniform tumor progression, follows a clinical progression of the disease, and requires minimal genetic alterations in mice with high potential to advance the preclinical testing of new therapeutics.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Oral facial cleft (OFC) comprises cleft lip with or without cleft palate (CL/P) or cleft palate only.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) in many genomic loci where the presumed effector gene (for example, IRF6 in the 1q32 locus) is expressed in embryonic oral epithelium.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
To identify candidates for functional SNPs at eight such loci we conduct a massively parallel reporter assay in a fetal oral epithelial cell line, revealing SNPs with allele-specific effects on enhancer activity.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
We filter these SNPs against chromatin-mark evidence of enhancers and test a subset in traditional reporter assays, which support the candidacy of SNPs at loci containing FOXE1, IRF6, MAFB, TFAP2A, and TP63.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
For two SNPs near IRF6 and one near FOXE1, we engineer the genome of induced pluripotent stem cells, differentiate the cells into embryonic oral epithelium, and discover allele-specific effects on the levels of effector gene expression, and, in two cases, the binding affinity of transcription factors FOXE1 or ETS2.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Conditional analyses of GWAS data suggest the two functional SNPs near IRF6 account for the majority of risk for CL/P at this locus.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
This study connects genetic variation associated with OFC to mechanisms of pathogenesis.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Oral facial cleft (OFC) is a multifactorial disorder that can present as a cleft lip with or without cleft palate (CL/P) or a cleft palate only (CP) and has a genetic predisposition.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
More than one hundred syndromes include OFC as a phenotype, and overall, about 30% of CL/P cases are syndromic.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Such syndromes are generally caused by mutations in single genes and follow a Mendelian inheritance pattern.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
The remaining cases are non-syndromic or isolated.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
The etiology of non-syndromic OFC is partially genetic, as the concordance of non-syndromic OFC is 50% in identical twins but just 3–5% in other first-degree relatives.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Multiple independent genome-wide association studies (GWAS), and meta-analyses thereof, have identified more than 40 loci where alleles of common single-nucleotide polymorphisms (SNPs) are over-represented in cases versus in controls with the same ancestry (reviewed in ref. ).
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Importantly, however, most of the heritable risk for isolated OFC has not been assigned to any gene or locus.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Moreover, GWAS results alone do not illuminate the mechanisms of pathogenesis attributable to genetic variation at each locus.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Understanding these mechanisms may point to additional genes in which mutations will contribute risk for non-syndromic OFC and may guide the design of preventative therapies.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Identifying functional (causal) SNPs among those in linkage disequilibrium with them is challenging.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Most SNPs lie in non-coding DNA, and the functional subset of them presumably disrupt cis-regulatory sequences (i.e., enhancers and promoters).
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
However, the sequence constraints of cis-regulatory sequences remain poorly understood.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
We and others have used machine learning to mine sets of tissue-specific enhancers for sequence patterns, and there are in silico tools for inferring variants that affect enhancer function, but currently there is no tool that can robustly identify non-coding variants that alter enhancer activity.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
An alternative approach is the massively parallel reporter assay (MPRA).
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
MPRAs have been widely used to detect elements with enhancer activity, and in some cases to detect the effect of variants on enhancer activity.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
A challenge in deploying MPRA to identify functional SNPs is that, because the results are dependent on the cellular context, it may be necessary to use a cell line that models the embryonic cell type in which the SNPs affect disease risk.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
The 1q32/IRF6 locus is associated with OFC in multiple ethnic groups.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
IRF6, encoding the transcription factor Interferon Regulatory Factor 6, is strong candidate for the risk-relevant gene (i.e., the effector gene) in this locus because mutations in IRF6 are found in about 70% of patients with Van der Woude syndrome, the most common syndromic form of OFC (VWS1, OMIM # 119300).
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Finding the functional SNPs at this locus would yield insight into the regulation of IRF6 gene expression during morphogenesis of the face and, if the transcription factors whose binding is affected by those SNPs are identified, would identify new candidate genes for OFC.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
However, a meta-analysis of several GWAS identified more than 600 SNPs at this locus with P values indicating at least a suggestive association with OFC.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
One of these SNPs, rs642961, resides in an enhancer of IRF6 and alters the binding of the transcription factor AP2-α (TFAP2A) in an electrophoretic mobility shift assay, suggesting it is functional.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
However, this conclusion is uncertain because this SNP did not have allele-specific effects in a standard reporter assay or in an MPRA.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
The large number of OFC-associated SNPs at this locus, and others, makes it difficult to determine which are functional.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Here, we deployed an MPRA in a fetal oral epithelium cell line to nominate candidate functional SNPs among those associated with OFC and within loci where the presumed effector gene is expressed in oral epithelium.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
We validated a subset of the MPRA results using traditional luciferase reporter assays in the cell line and in primary keratinocytes.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
For two promising SNPs near IRF6 and one near FOXE1, we engineered the genotype of the SNPs in induced pluripotent stem cells, differentiated the cells into embryonic oral epithelium, and then assessed allele-specific effects on gene expression and transcription factor binding.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
These studies support six SNPs as being functional, with varying levels of support, and two of these SNPs as accounting for most of the heritable risk for CL/P phenotype attributed to the IRF6 locus in the cohort analyzed here.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
For analysis by MPRA, we picked 887 SNPs from eight loci associated with OFC and in which the currently presumed effector gene is expressed in oral epithelium, although not necessarily only there, and which regulates differentiation of an epithelial tissue (1q32/IRF6, 2p21/THADA, 3q28/TP63, 6p24.3/TFAP2A, 9q22.2/GADD45G, 9q22.33/FOXE1, 12q13.13/KRT18, and 20q12/MAFB) (Table 1 and Supplementary Data 1, 2).
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
At seven of the loci, we picked SNPs based on their significance in a genome-wide meta-analysis of two prior OFC studies (Table 1 and Supplementary Data 2).
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
We additionally included SNPs at the TFAP2A locus identified in an independent GWAS of CL/P in Han Chinese.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
At an eighth locus (9q22.33/FOXE1), we picked SNPs in strong linkage disequilibrium with the GWAS lead SNP (rs12347191) and annotated as being within regulatory elements (Table 1 and Supplementary Data 2).
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
As outlined in Fig. 1a, we synthesized a library of reporter plasmids containing 161 base-pairs (bp) genomic test elements each centered on an OFC-associated SNP; OFC risk and non-risk alleles of each SNP were represented in four replicate constructs with distinct barcodes (Supplementary Data 3, 4).
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
We performed the MPRA in GMSM-K cells (Supplementary Data 5), a cell line derived from human fetal oral mucosa.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
The results were strongly correlated (R ≥ 0.876) across four replicate experiments (Supplementary Fig. 1a).Fig.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
1MPRA identified SNPs with allele-specific significant effects on reporter activity.a Schematic of MPRA library construction and execution.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
BC, barcode.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Yellow and red asterisks: non-risk and risk allele, respectively.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
b Scatter dot plot showing (black dots) 65 SNPs with significant allele-specific effects on reporter activity in the MPRA and (gray dots) 822 SNPs without them.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Arrows indicate the functional SNPs identified in this study (two near IRF6, rs11119348 and rs661849, and one near FOXE1, rs10984103).
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Dashed lines indicate the 95th percentile of the reporter activity of scrambled elements; on both axes, zero, i.e., log2(1) represents the reporter activity of the empty vector.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
Table 1The number of SNPs tested, and the number with allele-specific effects, in the MPRA at each locusLocusTested in MPRA (887 total)Allele-specific effects in MPRA (65 total)Further supported by chromatin evidence in embryonic faces and/or NHEKTested in luciferase reporter assays in HEKn and GMSM-KTested by genome engineering in induced oral epithelium1q32/IRF6608469rs11119348, rs661849rs11119348, rs6618492p21/THADA401-3q28/TP631411rs754368776p24.3/TFAP2A11282rs2012659q22.2/GADD45G362-9q22.33/FOXE1911rs10984103rs1098410312q13.13/KRT18142-20q12/MAFB5441rs4812449 a Schematic of MPRA library construction and execution.
PMC12267437
Identification of functional non-coding variants associated with orofacial cleft
BC, barcode.