PRMT1 Inhibition Selectively Targets BNC1-Dependent Proliferation, but not Migration in Squamous Cell Carcinoma.

Squamous Cell Carcinoma (SCC) develops in stratified epithelial tissues and demonstrates frequent alterations in transcriptional regulators. We sought to discover SCC-specific transcriptional programs and identified the transcription factor Basonuclin 1 (BNC1) as highly expressed in SCC compared to other tumor types. RNA-seq and ChIP-seq analysis identified pro-proliferative genes activated by BNC1 in SCC cells and keratinocytes. Inhibition of BNC1 in SCC cells suppressed proliferation and increased migration via FRA1. In contrast, BNC1 reduction in keratinocytes caused differentiation, which was abrogated by IRF6 knockdown, leading to increased migration. Protein interactome analysis identified PRMT1 as a co-activator of BNC1-dependent proliferative genes. Inhibition of PRMT1 resulted in a dose-dependent reduction in SCC cell proliferation without increasing migration. Importantly, therapeutic inhibition of PRMT1 in SCC xenografts significantly reduced tumor size, resembling functional effects of BNC1 knockdown. Together, we identify BNC1-PRMT1 as an SCC-lineage specific transcriptional axis that promotes cancer growth, which can be therapeutically targeted to inhibit SCC tumorigenesis.

Home Spirometry in Children with Cystic Fibrosis.

We report the implementation of a pediatric home spirometry program at our institution. A respiratory therapist provided either a virtual or an in-person initiation visit that included a coached spirometry session. Families were instructed to perform daily uncoached spirometry sessions for 5 days. The program's quality assurance component was deemed not to be human research by the local IRB. In total, 52 subjects completed an initiation visit (34 with at least 3 additional uncoached spirometry sessions). The clinic spirometry and coached (same-day) sessions and uncoached (same-week) sessions were completed by 12 and 17 subjects, respectively. The median (99% CI) coefficients of variation for FEV1% of the uncoached maneuvers were 3.5% (2.9-5.9%). The median (IQR) FEV1% and FEV1 (mL) absolute differences between coached and uncoached home spirometry were -2% (-4 and +3%) and -25 mL (-93 and +93 mL), respectively. The median (IQR) absolute differences in FEV1% and FEV1 (mL) between coached or uncoached home spirometry and clinic spirometry were -6% (-10 and -2%) and -155 mL (-275 and -88 mL), and -4% (-10 and +5%), and -110 mL (-280 and +9 mL), respectively. Differences in absolute FEV1 (L) and FEV1% were found among different modalities of spirometry performed by people with cystic fibrosis. Understanding the variability of uncoached home spirometry and the differences among coached and uncoached home spirometry, hospital and coached home spirometry, and hospital and uncoached home spirometry for any given individual is crucial to effectively utilize this tool in clinical care.

IL1α Antagonizes IL1ß and Promotes Adaptive Immune Rejection of Malignant Tumors.

We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1ß-/-, IL1α-/-, and IL1R1-/- mice. Tumors grew progressively in IL1R-/- and IL1α-/- mice but were often absent in IL1ß-/- mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1ß prevented tumor growth in wild-type (WT) mice but not in IL1R1-/- or IL1α-/- mice. Antibodies to IL1α promoted tumor growth in IL1ß-/- mice and reversed the tumor-suppressive effect of anti-IL1ß in WT mice. Depletion of CD8+ T cells and blockade of lymphocyte mobilization abrogated the IL1ß-/- tumor suppressive effect, as did crossing IL1ß-/- mice to SCID or Rag1-/- mice. Finally, blockade of IL1ß synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1ß promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell-mediated antitumor immunity.

Loss of GCNT2/I-branched glycans enhances melanoma growth and survival.

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.

Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans.

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-ß1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.

IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

Development of enantioselective synthetic routes to the hasubanan and acutumine alkaloids.

We describe a general strategy to prepare the hasubanan and acutumine alkaloids, a large family of botanical natural products that display antitumor, antiviral, and memory-enhancing effects. The absolute stereochemistry of the targets is established by an enantioselective Diels-Alder reaction between 5-(trimethylsilyl)cyclopentadiene (36) and 5-(2-azidoethyl)-2,3-dimethoxybenzoquinone (24). The Diels-Alder adduct 38 is transformed to the tetracyclic imine 39 by a Staudinger reduction-aza-Wittig sequence. The latter serves as a universal precursor to the targets. Key carbon-carbon bond constructions include highly diastereoselective acetylide additions to the N-methyliminium ion derived from 39 and Friedel-Crafts and Hosomi-Sakurai cyclizations to construct the carbocyclic skeleton of the targets. Initially, this strategy was applied to the syntheses of (-)-acutumine (4), (-)-dechloroacutumine (5), and four hasubanan alkaloids (1, 2, 3, and 8). Herein, the synthetic route is adapted to the syntheses of six additional hasubanan alkaloids (12, 13, 14, 15, 18, and 19). The strategic advantage of 5-(trimethylsilyl)cyclopentadiene Diels-Alder adducts is demonstrated by site-selective functionalization of distal carbon-carbon π-bonds in the presence of an otherwise reactive norbornene substructure. Evaluation of the antiproliferative properties of the synthetic metabolites revealed that four hasubanan alkaloids are submicromolar inhibitors of the N87 cell line.

Disruption of TNF-α/TNFR1 function in resident skin cells impairs host immune response against cutaneous vaccinia virus infection.

One strategy adopted by vaccinia virus (VV) to evade the host immune system is to encode homologs of TNF receptors (TNFRs) that block TNF-α function. The response to VV skin infection under conditions of TNF-α deficiency, however, has not been reported. We found that TNFR1-/- mice developed larger primary lesions, numerous satellite lesions, and higher skin virus levels after VV scarification. Following their recovery, VV-scarified TNFR1-/- mice were fully protected against challenge with a lethal intranasal dose of VV, suggesting these mice had developed an effective memory immune response. A functional systemic immune response was further demonstrated by enhanced production of VV-specific IFN-γ and VV-specific CD8(+) T cells in spleens and draining lymph nodes. Interestingly, bone marrow (BM)-reconstitution studies using wild-type (WT) BM in TNFR1-/- host mice, but not TNFR1-/- BM in WT host mice, reproduced the original results seen in TNFR1-/- mice, indicating that TNFR1 deficiency in resident skin cells, rather than hematopoietic cells, accounts for the impaired cutaneous immune response. Our data suggest that lack of TNFR1 leads to a skin-specific immune deficiency, and that resident skin cells have a crucial role in mediating an optimal immune defense to VV cutaneous infection via TNF-α/TNFR1 signaling.

CD43 is a ligand for E-selectin on CLA+ human T cells.

The recruitment of memory T cells from blood into tissues is a central element of immune surveillance and adaptive immune responses and a key feature of chronic cutaneous inflammatory diseases such as psoriasis and atopic dermatitis. Human memory T cells that infiltrate skin express the carbohydrate epitope cutaneous lymphocyte-associated antigen (CLA). Expression of the CLA epitope on T cells has been described on P-selectin glycoprotein ligand-1 (PSGL-1) and associated with the acquisition of both E-selectin and P-selectin ligand functions. In this report, we show that CD43, a sialomucin expressed constitutively on T cells, can also be decorated with the CLA epitope and serve as an E-selectin ligand. CLA expressed on CD43 was found exclusively on the high-molecular-weight (125 kDa) glycoform bearing core-2-branched O-linked glycans. CLA+ CD43 purified from human T cells supported tethering and rolling in shear flow via E-selectin but did not support binding of P-selectin. The identification and characterization of CD43 as a T-cell E-selectin ligand distinct from PSGL-1 expands the role of CD43 in the regulation of T-cell trafficking and provides new targets for the modulation of immune functions in skin.

Direct real-time observation of E- and P-selectin-mediated rolling on cutaneous lymphocyte-associated antigen immobilized on Western blots.

Human memory T cells associated with cutaneous inflammatory responses are characterized by their expression of cutaneous lymphocyte-associated Ag (CLA), a carbohydrate determinant differentially expressed on P-selectin glycoprotein ligand-1 (PSGL-1). Although expression of the CLA epitope on PSGL-1 (CLA(+) PSGL-1) by memory T cells is associated with acquisition of E-selectin ligand activity, it is not known whether CLA(+) PSGL-1, itself, is a ligand for E-selectin on human T cells or whether other glycoproteins, with or without CLA modification, support E-selectin-dependent rolling in shear flow. To address this issue, we developed a method for real-time analysis of functional adhesive interactions between selectin-bearing cells in shear flow with leukocyte ligands resolved by SDS-PAGE and immobilized on standard Western blots. The results of these studies provide direct evidence that CLA(+) PSGL-1 is a functional ligand for both E- and P-selectin, confirm that the P-selectin ligand activity of PSGL-1 is independent of CLA modification, and identify a distinct, non-PSGL-1 E-selectin ligand on CLA-positive human memory T cells.