UHRF2 regulates cell cycle, epigenetics and gene expression to control the timing of retinal progenitor and ganglion cell differentiation.

Ubiquitin-like, containing PHD and RING finger domains 2 (UHRF2) regulates cell cycle and binds 5-hydroxymethylcytosine (5hmC) to promote completion of DNA demethylation. Uhrf2-/- mice are without gross phenotypic defects; however, the cell cycle and epigenetic regulatory functions of Uhrf2 during retinal tissue development are unclear. Retinal progenitor cells (RPCs) produce all retinal neurons and Müller glia in a predictable sequence controlled by the complex interplay between extrinsic signaling, cell cycle, epigenetic changes and cell-specific transcription factor activation. In this study, we find that UHRF2 accumulates in RPCs, and its conditional deletion from mouse RPCs reduced 5hmC, altered gene expressions and disrupted retinal cell proliferation and differentiation. Retinal ganglion cells were overproduced in Uhrf2-deficient retinae at the expense of VSX2+ RPCs. Most other cell types were transiently delayed in differentiation. Expression of each member of the Tet3/Uhrf2/Tdg active demethylation pathway was reduced in Uhrf2-deficient retinae, consistent with locally reduced 5hmC in their gene bodies. This study highlights a novel role of UHRF2 in controlling the transition from RPCs to differentiated cell by regulating cell cycle, epigenetic and gene expression decisions.

Chondroitin sulfate proteoglycan 4 (CSPG4) increases invasion of recessive dystrophic epidermolysis bullosa-associated cutaneous squamous cell carcinoma by modifying TGFß signaling.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic skin-blistering disorder often progressing to metastatic cutaneous squamous cell carcinoma (cSCC) at chronic wound sites. Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell-surface proteoglycan that is an oncoantigen in multiple malignancies, where it modulates oncogenic signaling, drives epithelial-to-mesenchymal transition (EMT), and enables cell motility. OBJECTIVES: To evaluate CSPG4 expression and function in RDEB-cSCC. METHODS: RDEB-cSCC cell lines were used to assess CSPG4-dependent changes in invasive potential, TGFß1-stimulated signal activation, and clinically relevant cytopathology metrics in an in vitro full-thickness tumor model. CSPG4 expression in RDEB-cSCC and non-RDEB cSCC tumors was analyzed via immunohistochemistry and single-cell RNA sequencing (scRNA-seq), respectively. RESULTS: Inhibiting CSPG4 expression reduced invasive potential in multiple RDEB-cSCC cell lines and altered membrane-proximal TGFß signal activation through changes in SMAD3 phosphorylation. CSPG4 expression was uniformly localized to basal-layer keratinocytes in fibrotic RDEB skin and tumor cells at the tumor/stroma interface at the invasive front in RDEB-cSCC tumors in vivo. Analysis of published scRNA-seq data revealed that CSPG4 expression was correlated with an enhanced EMT transcriptomic signature in cells at the tumor/stroma interface of non-RDEB cSCC tumors. Cytopathological metrics, like nucleus:cell area ratio, were influenced by CSPG4 expression in in vitro tumor models. CONCLUSIONS: We determined that CSPG4 expression in RDEB-cSCC cell lines enhanced invasive potential. Mechanistically, CSPG4 was found to enhance membrane-proximal TGFß-stimulated signaling through SMAD3, which is a key mediator of EMT in RDEB-cSCC. The implication of these studies is that CSPG4 may represent a therapeutic target that can be leveraged for clinical management in patients with RDEB-cSCC.

Receptor for hyaluronan-mediated motility (RHAMM) defines an invasive niche associated with tumor progression and predicts poor outcomes in breast cancer patients.

Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant synthesis, processing, and signaling of hyaluronan (HA). Hyaluronan-mediated motility receptor (RHAMM, CD168; HMMR) is an HA receptor enabling tumor cells to sense and respond to this aberrant TME during breast cancer progression. Previous studies have associated RHAMM expression with breast tumor progression; however, cause and effect mechanisms are incompletely established. Focused gene expression analysis of an internal breast cancer patient cohort confirmed that increased RHAMM expression correlates with aggressive clinicopathological features. To probe mechanisms, we developed a novel 27-gene RHAMM-related signature (RRS) by intersecting differentially expressed genes in lymph node (LN)-positive patient cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validated in an independent cohort. We demonstrate that the RRS predicts for poor survival and is enriched for cell cycle and TME-interaction pathways. Further analyses using CRISPR/Cas9-generated RHAMM-/- breast cancer cells provided direct evidence that RHAMM promotes invasion in vitro and in vivo. Immunohistochemistry studies highlighted heterogeneous RHAMM protein expression, and spatial transcriptomics associated the RRS with RHAMM-high microanatomic foci. We conclude that RHAMM upregulation leads to the formation of 'invasive niches', which are enriched in RRS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Blocking Site-Specific Cleavage of Human Tau Delays Progression of Disease-Related Phenotypes in Genetically Matched Tau-Transgenic Mice Modeling Frontotemporal Dementia.

Studies have recently demonstrated that a caspase-2-mediated cleavage of human tau (htau) at asparate-314 (D314) is responsible for cognitive deficits and neurodegeneration in mice modeling frontotemporal dementia (FTD). However, these animal studies may be confounded by flaws in their model systems, such as endogenous functional gene disruption and inequivalent transgene expression. To avoid these weaknesses, we examined the pathogenic role of this site-specific htau cleavage in FTD using genetically matched htau targeted-insertion mouse lines: rT2 and rT3. Both male and female mice were included in this study. rT2 mice contain a single copy of the FTD-linked htau proline-to-leucine mutation at amino acid 301 (htau P301L), inserted into a neutral site to avoid dysregulation of host gene expression. The similarly constructed rT3 mice harbor an additional D314-to-glutamate (D314E) mutation that blocks htau cleavage. We demonstrate that htau transgene expression occurs primarily in the forebrain at similar levels in rT2 and rT3 mice. Importantly, expression of the cleavage-resistant D314E mutant delays transgene-induced tau accumulation in the postsynaptic density, brain atrophy, hippocampal neurodegeneration, and spatial memory impairment, without altering age-related progression of pathologic tau conformation and phosphorylation. Our comprehensive investigation of age-dependent disease phenotypes associated with the htau P301L variant in precisely engineered FTD-modeling mice unveils a transiently protective effect of blocking htau cleavage at D314. Findings of this study advance our understanding of the contribution of this tau cleavage to the pathogenesis of FTD, and aid the development of effective dementia-targeting therapies.SIGNIFICANCE STATEMENT A site-specific and caspase-2-mediated cleavage of human tau plays a pathologic role in dementia. In this study, we investigate the contribution of this cleavage to the pathogenesis of frontotemporal dementia (FTD) using two genetically matched, tau-transgene targeted-insertion mouse lines that differ only by a cleavage-resistant mutation. The use of these mice avoids confounding effects associated with the random integration of tau transgenes to the mouse genome and allows us to comprehensively evaluate the impact of the tau cleavage on FTD phenotypes. Our data reveal that blocking this tau cleavage delays memory impairment and neurodegeneration of FTD-modeling mice. These findings improve our understanding of the pathogenic mechanisms underlying FTD and will facilitate the development of effective therapeutics.

Retinoblastoma tumor cell proliferation is negatively associated with an immune gene expression signature and increased immune cells.

This study focuses on gene expression differences between early retinal states that ultimately lead to normal development, late onset retinoblastoma, or rapid bilateral retinoblastoma tumors. The late-onset and early-onset retinoblastoma tumor cells are remarkably similar to normally proliferating retinal progenitor cells, but they fail to properly express differentiation markers associated with normal development. Further, early-onset retinoblastoma tumor cells express a robust immune gene expression signature followed by accumulation of dendritic, monocyte, macrophage, and T-lymphocyte cells in the retinoblastoma tumors. This characteristic was not shared by either normal retinae or late-onset retinoblastomas. Comparison of our data with other human and mouse retinoblastoma tumor gene expression significantly confirmed, that the immune signature is present in tumors from each species. Strikingly, we observed that the immune signature in both mouse and human tumors was most highly evident in those with the lowest proliferative capacity. We directly assessed this relationship in human retinoblastoma tumors by co-analyzing proliferation and immune cell recruitment by immunohistochemistry, uncovering a significant inverse relationship between increased immune-cell infiltration in tumors and reduced tumor cell proliferation. Directly inhibiting proliferation with a PI3K/mTOR inhibitor significantly increased the number of CD45+ immune cells in the retina. This work establishes an in vivo model for the rapid recruitment of immune cells to tumorigenic neural tissue.

The identification of a novel antibody for CD133 using human antibody phage display.

BACKGROUND: The transmembrane glycoprotein CD133 is believed to be a marker of adult prostate stem cells and cancer stem/initiating cells. Investigating the role of CD133 in the normal biology of the prostate and in cancer is complicated by the lack of a sensitive and accurate antibody for its detection. Here, we describe the characterization of a unique antibody identified using human antibody phage display that can recognize CD133 in both formalin-fixed tissues and cell lines. METHODS: A human single-chain variable fragment (scFv) antibody phage display library possessing a diversity of 8 × 109 was screened against fully glycosylated recombinant CD133. A counter screen was performed against deglycosylated CD133 to select for clones that preferentially recognized a glycosylation-independent epitope. The lead scFv was analyzed by flow cytometry and cloned into a rabbit immunoglobulin scaffold for immunohistochemistry (IHC). RESULTS: The antibody designated HA10 was found to bind a glycosylation-independent epitope on the peptide backbone of CD133 with high affinity. As a reagent for flow cytometry, HA10 detected CD133 more accurately than a commonly used commercially available antibody. IHC analysis with HA10 documented the staining of basal cells and luminal cells in healthy prostate sections. Weak staining of luminal cells was observed in adenocarcinoma sections at a very low frequency. Examination of a LuCaP patient-derived xenograft tissue microarray found that only three of the LuCaP models were positive for CD133. The three CD133pos LuCaP models all originated from non-AR driven metastatic prostate cancer with neuroendocrine differentiation. Subsequent interrogation of liver biopsies from a patient who failed second-generation anti-androgen therapy found high levels of CD133 staining. The original transurethral resection of the prostate from that patient was, however, absent of CD133. CONCLUSIONS: We have developed a novel antibody that was able to detect CD133 by both IHC and flow cytometry. Using HA10 as an IHC reagent, we found that CD133 is a marker for a very rare cell type in both healthy prostate and adenocarcinoma sections. Our preliminary investigation also suggests that there may be an association between CD133 and non-AR driven prostate cancer with neuroendocrine differentiation.

Mesenchymal stromal cells inhibit murine syngeneic anti-tumor immune responses by attenuating inflammation and reorganizing the tumor microenvironment.

The potential of mesenchymal stromal cells (MSCs) to inhibit anti-tumor immunity is becoming increasingly well recognized, but the precise steps affected by these cells during the development of an anti-tumor immune response remain incompletely understood. Here, we examined how MSCs affect the steps required to mount an effective anti-tumor immune response following administration of adenovirus Fas ligand (Ad-FasL) in the Lewis lung carcinoma (LL3) model. Administration of bone marrow-derived MSCs with LL3 cells accelerated tumor growth significantly. MSCs inhibited the inflammation induced by Ad-FasL in the primary tumors, precluding their rejection; MSCs also reduced the consequent expansion of tumor-specific T cells in the treated hosts. When immune T cells were transferred to adoptive recipients, MSCs impaired, but did not completely abrogate the ability of these T cells to promote elimination of secondary tumors. This impairment was associated with a modest reduction in tumor-infiltrating T cells, with a significant reduction in tumor-infiltrating macrophages, and with a reorganization of the stromal environment. Our data indicate that MSCs in the tumor environment reduce the efficacy of immunotherapy by creating a functional and anatomic barrier that impairs inflammation, T cell priming and expansion, and T cell function-including recruitment of effector cells.

Trp53 haploinsufficiency modifies EGFR-driven peripheral nerve sheath tumorigenesis.

Malignant peripheral nerve sheath tumors (MPNSTs) are genetically diverse, aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 1 syndrome. Reduced TP53 gene expression and amplification/overexpression of the epidermal growth factor receptor (EGFR) gene occur in MPNST formation. We focused on determining the cooperativity between reduced TP53 expression and EGFR overexpression for Schwann cell transformation in vitro (immortalized human Schwann cells) and MPNST formation in vivo (transgenic mice). Human gene copy number alteration data, microarray expression data, and TMA analysis indicate that TP53 haploinsufficiency and increased EGFR expression co-occur in human MPNST samples. Concurrent modulation of EGFR and TP53 expression in HSC1λ cells significantly increased proliferation and anchorage-independent growth in vitro. Transgenic mice heterozygous for a Trp53-null allele and overexpressing EGFR in Schwann cells had a significant increase in neurofibroma and grade 3 PNST (MPNST) formation compared with single transgenic controls. Histological analysis of tumors identified a significant increase in pAkt expression in grade 3 PNSTs compared with neurofibromas. Array comparative genome hybridization analysis of grade 3 PNSTs identified recurrent focal regions of chromosomal gains with significant enrichment in genes involved in extracellular signal-regulated kinase 5 signaling. Collectively, altered p53 expression cooperates with overexpression of EGFR in Schwann cells to enhance in vitro oncogenic properties and tumorigenesis and progression in vivo.

Three-Dimensional Cell Entrapment as a Function of the Weight Percent of Peptide-Amphiphile Hydrogels.

The design of scaffolds which mimic the stiffness, nanofiber structure, and biochemistry of the native extracellular matrix (ECM) has been a major objective for the tissue engineering field. Furthermore, mimicking the innate three-dimensional (3D) environment of the ECM has been shown to significantly altered cellular response compared to that of traditional two-dimensional (2D) culture. We report the development of a self-assembling, fibronectin-mimetic, peptide-amphiphile nanofiber scaffold for 3D cell culture. To form such a scaffold, 5 mol % of a bioactive PR_g fibronectin-mimetic peptide-amphiphile was mixed with 95 mol % of a diluent peptide-amphiphile (E2) whose purpose was to neutralize electrostatic interactions, increase the gelation kinetics, and promote cell survival. Atomic force microscopy verified the fibrilar structure of the gels, and the mechanical properties were characterized for various weight percent (wt %) formulations of the 5 mol % PR_g-95 mol % E2 peptide-amphiphile mixture. The 0.5 wt % formulations had an elastic modulus of 429.0 ± 21.3 Pa whereas the 1.0 wt % peptide-amphiphile hydrogels had an elastic modulus of 808.6 ± 38.1 Pa. The presence of entrapped cells in the gels decreased the elastic modulus, and the decrease was a function of cell loading. Although both formulations supported cell proliferation, the 0.5 wt % gels supported significantly greater NIH3T3/GFP fibroblast cell proliferation throughout the gels than the 1.0 wt % gels. However, compared to the 0.5 wt % formulations, the 1.0 wt % hydrogels promoted greater increases in mRNA expression and the production of fibronectin and type IV collagen ECM proteins. This study suggests that this fibronectin-mimetic scaffold holds great promise in the advancement of 3D culture applications and cell therapies.

Elevated hyaluronan and hyaluronan-mediated motility receptor are associated with biochemical failure in patients with intermediate-grade prostate tumors.

BACKGROUND: The clinical course of prostate cancer (PCa) measured by biochemical failure (BF) after prostatectomy remains unpredictable in many patients, particularly in intermediate Gleason score (GS) 7 tumors, suggesting that identification of molecular mechanisms associated with aggressive PCa biology may be exploited for improved prognostication or therapy. Hyaluronan (HA) is a high molecular weight polyanionic carbohydrate produced by synthases (HAS1 through HAS3) and fragmented by oxidative/nitrosative stress and hyaluronidases (HYAL1 through HYAL4, SPAM1) common in PCa microenvironments. HA and HA fragments interact with receptors CD44 and hyaluronan-mediated motility receptor (HMMR), resulting in increased tumor aggressiveness in experimental PCa models. This study evaluated the association of HA-related molecules with BF after prostatectomy in GS7 tumors. METHODS: Tissue microarrays were constructed from a 96-patient cohort. HA histochemistry and HAS2, HYAL1, CD44, CD44v6, and HMMR immunohistochemistry were quantified using digital pathology techniques. RESULTS: HA in tumor-associated stroma and HMMR in malignant epithelium were significantly and marginally significantly associated with time to BF in univariate analysis, respectively. After adjusting for clinicopathologic features, both HA in tumor-associated stroma and HMMR in malignant epithelium were significantly associated with time to BF. Although not significantly associated with BF, HAS2 and HYAL1 positively correlated with HMMR in malignant epithelium. Cell culture assays demonstrated that HMMR bound native and fragmented HA, promoted HA uptake, and was required for a promigratory response to fragmented HA. CONCLUSIONS: HA and HMMR are factors associated with time to BF in GS7 tumors, suggesting that increased HA synthesis and fragmentation within the tumor microenvironment stimulates aggressive PCa behavior through HA-HMMR signaling.

Evaluation of protein biomarkers of prostate cancer aggressiveness.

BACKGROUND: Prognostic multibiomarker signatures in prostate cancer (PCa) may improve patient management and provide a bridge for developing novel therapeutics and imaging methods. Our objective was to evaluate the association between expression of 33 candidate protein biomarkers and time to biochemical failure (BF) after prostatectomy. METHODS: PCa tissue microarrays were constructed representing 160 patients for whom clinicopathologic features and follow-up data after surgery were available. Immunohistochemistry for each of 33 proteins was quantified using automated digital pathology techniques. Relationships between clinicopathologic features, staining intensity, and time to BF were assessed. Predictive modeling using multiple imputed datasets was performed to identify the top biomarker candidates. RESULTS: In univariate analyses, lymph node positivity, surgical margin positivity, non-localized tumor, age at prostatectomy, and biomarkers CCND1, HMMR, IGF1, MKI67, SIAH2, and SMAD4 in malignant epithelium were significantly associated with time to BF. HMMR, IGF1, and SMAD4 remained significantly associated with BF after adjusting for clinicopathologic features while additional associations were observed for HOXC6 and MAP4K4 following adjustment. In multibiomarker predictive models, 3 proteins including HMMR, SIAH2, and SMAD4 were consistently represented among the top 2, 3, 4, and 5 most predictive biomarkers, and a signature comprised of these proteins best predicted BF at 3 and 5 years. CONCLUSIONS: This study provides rationale for investigation of HMMR, HOXC6, IGF1, MAP4K4, SIAH2, and SMAD4 as biomarkers of PCa aggressiveness in larger cohorts.

Digital and manual interfollicular Ki-67 are associated with a progression-free survival in patients with low-grade follicular lymphoma.

OBJECTIVES: Novel histopathologic prognostic factors are needed to identify patients with follicular lymphoma (FL) at risk of inferior outcomes. Our primary objective was to evaluate the Ki-67 proliferative index in follicular and interfollicular areas in tissue biopsy specimens from patients with newly diagnosed FL and correlate with clinical outcomes. Our secondary objective was to correlate PD-L1 and LAG-3 with clinical outcomes. METHODS: Seventy cases of low-grade FL from the University of Minnesota were evaluated with Ki-67 immunohistochemical stain. Ki-67 expression as a continuous variable was interpreted digitally and manually in follicular and interfollicular areas. Progression-free survival (PFS) and overall survival (OS) were analyzed by Cox regression, and hazard ratios (HRs) per 10-point increase in Ki-67 were calculated. RESULTS: Progression-free survival at 4 years was 28% (95% CI, 19%-41%). Interfollicular, but not follicular, Ki-67 was associated with PFS by manual (HR, 1.33; P = .01) and digital (HR, 1.38; P = .02) analysis. Digital and manual Ki-67 were only moderately correlated but demonstrated similar effects on PFS. At 4 years, OS was 90% with no association with follicular or interfollicular Ki-67 proliferation. CONCLUSIONS: Higher interfollicular Ki-67 by either digital or manual analysis is associated with a poorer PFS in patients with low-grade FL. These results suggest further validation of this marker is warranted to improve pathologic risk stratification at FL diagnosis. PD-L1 and LAG-3 were not associated with PFS or OS.

Hemangiosarcoma Cells Promote Conserved Host-derived Hematopoietic Expansion.

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors. SIGNIFICANCE: We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation, providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression.

Genomically Complex Human Angiosarcoma and Canine Hemangiosarcoma Establish Convergent Angiogenic Transcriptional Programs Driven by Novel Gene Fusions.

Sporadic angiosarcomas are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic angiosarcomas. In this study, we leveraged RNA-sequencing data from 13 human angiosarcomas and 76 spontaneous canine hemangiosarcomas to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in seven of the 13 human tumors, with two tumors showing mutations of TP53. HRAS and NRAS mutations were found in angiosarcomas without fusions or TP53 mutations. We found 15 novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 in 11 of the 76 canine hemangiosarcomas; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS. In particular, fusion genes and mutations of TP53 cooccurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human angiosarcomas and canine hemangiosarcomas identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data suggest that genome instability induced by TP53 mutations might create a predisposition for fusion events that may contribute to tumor progression by promoting selection and/or enhancing fitness through activation of convergent angiogenic pathways in this vascular malignancy. IMPLICATIONS: This study shows that, while drive events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphologic organization and biological behavior of these tumors in both species.

A Rabbit Monoclonal Antibody against the Antiviral and Cancer Genomic DNA Mutating Enzyme APOBEC3B.

The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease.

Rituximab-induced changes in hematolymphoid tissues found at autopsy.

The effect of rituximab on malignant B cells and normal circulating B cells has been previously studied. In contrast, data on the degree of depletion of nonneoplastic B cells induced by rituximab in lymph nodes and spleen is limited. For this purpose, clinical charts, autopsy records, lymph node and spleen sections, and immunoperoxidase stains were reviewed from 10 patients who had received 1 to 40 doses of rituximab before death. The percentage of nonneoplastic B cells was lower in the lymph node and spleen in rituximab-treated patients when compared with cyclophosphamide, doxorubicin, vincristine, and prednisone-treated patients and patients without lymphoma. The effect of rituximab on nonneoplastic B cells was observed as soon as 1 month after administration and with as few as 3 doses. Reappearance of normal numbers of B cells was not observed 1 to 12 months after the last dose of rituximab was administered. We conclude that rituximab induces prompt, consistent, profound, and prolonged depletion of B lymphocyte populations in human lymphoid tissue.

Retinoblastoma cells activate the AKT pathway and are vulnerable to the PI3K/mTOR inhibitor NVP-BEZ235.

Retinoblastoma is a pediatric cancer of the retina most often caused by inactivation of the retinoblastoma (RB1) tumor suppressor gene. We previously showed that Rb1 loss cooperates with either co-activating the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, or co-deleting Pten, to initiate retinoblastoma tumors in mice. The objectives of this study were to determine if the AKT pathway is activated in human retinoblastomas and the extent that anti-PI3K therapy induces apoptosis in retinoblastoma cells, alone or in combination with the DNA damaging drugs carboplatin and topotecan. Serial sections from human retinoblastoma tissue microarrays containing 27 tumors were stained with antibodies specific to p-AKT, Ki-67, forkhead box O1 (p-FOXO1), and ribosomal protein S6 (p-S6) using immunohistochemistry and each tumor sample scored for intensity. Human retinoblastoma tumors displayed significant correlation between p-AKT intensity with highly proliferative tumors (p = 0.008) that were also highly positive for p-FOXO1 (p = 0.002). Treatment with BEZ235, a dual PI3K/mTOR inhibitor, reduced phosphorylation levels of the AKT targets p-FOXO and p-S6 and effectively induced apoptosis the Y79 and Weri-1 human retinoblastoma cell lines and in vivo in our retinoblastoma mouse model. Long-term treatment with BEZ235 in vivo using our retinoblastoma-bearing mice induced apoptosis but did not significantly extend the lifespan of the mice. We then co-administered BEZ235 with topotecan and carboplatin chemotherapeutics in vivo, which more effectively induced apoptosis of retinoblastoma, but not normal retinal cells than either treatment alone. Our study has increased the variety of potentially effective targeted treatments that can be considered for human retinoblastoma.

Constitutive activation of alternative nuclear factor kappa B pathway in canine diffuse large B-cell lymphoma contributes to tumor cell survival and is a target of new adjuvant therapies.

Activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway is a common molecular event observed in both human and canine diffuse large B-cell lymphoma (DLBCL). Although the oncogenic potential of the alternative NFκB pathway (ANFκBP) has also been recently identified in DLBCL, its precise role in tumor pathogenesis and potential as a treatment target is understudied. We hypothesized that up-regulation of the ANFκBP plays an important role in the proliferation and survival of canine DLBCL cells, and we demonstrate that the ANFκBP is constitutively active in primary canine DLBCL samples and a cell line (CLBL1). We further demonstrate that a small interfering RNA inhibits the activation of the NFκB pathway and induces apoptosis in canine DLBCL cells. In conclusion, the ANFκBP facilitates survival of canine DLBCL cells, and thus, dogs with spontaneous DLBCL can provide a useful large animal model to study therapies targeting the ANFκBP.

Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-ß Protein Precursor Transgenic Mice.

There exist several dozen lines of transgenic mice that express human amyloid-ß protein precursor (AßPP) with Alzheimer's disease (AD)-linked mutations. AßPP transgenic mouse lines differ in the types and amounts of Aß that they generate and in their spatiotemporal patterns of expression of Aß assemblies, providing a toolkit to study Aß amyloidosis and the influence of Aß aggregation on brain function. More complete quantitative descriptions of the types of Aß assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aß toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AßPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ∼4.5 times that of 21-month-old Tg2576 mice and ∼15 times that of 21-24-month-old rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort.