Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth.

Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.

Molecular Profiles of Brain and Pulmonary Metastatic Disease in Cancer of Unknown Primary.

Cancers of unknown primary (CUP) are histologically confirmed malignancies but for which further investigation cannot identify a primary site. Improvements in histopathologic modalities for diagnosis have lessened the frequency of CUPs to 3%-5% of all malignancies compared with historical estimates of 5%-10%. Despite this, there is an ongoing debate as to whether CUPs are malignancies where the primary is not found or if they are otherwise a fully separate entity. Improvements in molecular analysis holds promise for improved identification and treatment of CUPs with mixed preliminary results. Here we present a woman with CUP and metastases in her brain and lung. We performed genomic profiling to compare the molecular makeup of each site in order to establish treatment targets. KEY POINTS: Cancer of unknown primary remains a diagnostic and therapeutic challenge. Molecular analysis may provide improvements in diagnosis and novel treatment options. Different sites of metastatic disease have subtle variations in molecular profile. Sequencing of different sites may offer therapeutic options that are either already approved or available in clinical trial.

Diffuse dermal angiomatosis mimicking inflammatory breast carcinoma.

Diffuse dermal angiomatosis (DDA) is a rare pathologically distinct subtype of reactive angioendotheliomatosis. In the literature, few biopsy-proven cases involving breast skin have been reported. We present a case of a 49-year-old female who presented with an indurated, erythematous, weeping, puckered and tender lesion with focal ulceration. Mammography demonstrated diffuse cutaneous and trabecular thickening concerning for inflammatory breast carcinoma. A punch biopsy demonstrated findings consistent with DDA. To our knowledge, this is the first reported case of DDA mimicking inflammatory carcinoma of the breast by clinical and radiologic examination.

Clinical features and treatment outcomes for primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder: a retrospective cohort study from the Dana-Farber Cancer Institute and updated literature review.

Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder (PCSM-TCLPD) was reclassified in 2016 as a rare benign entity with an excellent prognosis, yet its clinical features and best treatments remain poorly defined. We collected clinical data, treatments, and treatment-responses from our institution's patients with PCSM-TCLPD through September 2018 and an identical PubMed review through June 2021. Among 36 cases (median-age 54 years; 58.3% head/neck), diagnostic biopsy resulted in sustained complete remission (CR) in 13/33 punch/shave biopsies and 3/3 excisional biopsies. The remaining 20 patients further required topical corticosteroids (n = 5); intralesional corticosteroids (n = 1); surgical-excision (n = 5); electron-beam-radiation (n = 6); or brachytherapy (n = 3). All patients ultimately achieved CR, excluding one patient continuing treatment at end-of-study. 57/59 (96.6%) of institutional and literature-reported radiation-treated patients experienced CR. No institutional cases progressed beyond skin; 5/209 (2.4%) literature-reported cases progressed to systemic/extracutaneous involvement, all pre-reclassification. PCSM-TCLPD responds well to local-directed therapy including radiation, and only rarely if ever progresses.

Peripheral host T cells survive hematopoietic stem cell transplantation and promote graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating (Ki67+) and producing the proinflammatory cytokines IFN-γ and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling.

Double-stranded RNA (dsRNA), a frequent byproduct of virus infection, is recognized by Toll-like receptor 3 (TLR3) to mediate innate immune response to virus infection. TLR3 signaling activates the transcription factor IRF-3 by its Ser/Thr phosphorylation, accompanied by its dimerization and nuclear translocation. It has been reported that the Ser/Thr kinase TBK-1 is essential for TLR3-mediated activation and phosphorylation of IRF-3. Here we report that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways. One involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells. Thus, the PI3K-Akt pathway plays an essential role in TLR3-mediated gene induction.

High-throughput sequencing of the T cell receptor ß gene identifies aggressive early-stage mycosis fungoides.

Mycosis fungoides (MF), the most common cutaneous T cell lymphoma (CTCL) is a malignancy of skin-tropic memory T cells. Most MF cases present as early stage (stage I A/B, limited to the skin), and these patients typically have a chronic, indolent clinical course. However, a small subset of early-stage cases develop progressive and fatal disease. Because outcomes can be so different, early identification of this high-risk population is an urgent unmet clinical need. We evaluated the use of next-generation high-throughput DNA sequencing of the T cell receptor ß gene (TCRB) in lesional skin biopsies to predict progression and survival in a discovery cohort of 208 patients with CTCL (177 with MF) from a 15-year longitudinal observational clinical study. We compared these data to the results in an independent validation cohort of 101 CTCL patients (87 with MF). The tumor clone frequency (TCF) in lesional skin, measured by high-throughput sequencing of the TCRB gene, was an independent prognostic factor of both progression-free and overall survival in patients with CTCL and MF in particular. In early-stage patients, a TCF of >25% in the skin was a stronger predictor of progression than any other established prognostic factor (stage IB versus IA, presence of plaques, high blood lactate dehydrogenase concentration, large-cell transformation, or age). The TCF therefore may accurately predict disease progression in early-stage MF. Early identification of patients at high risk for progression could help identify candidates who may benefit from allogeneic hematopoietic stem cell transplantation before their disease becomes treatment-refractory.

Stat1 required for interferon-inducible but not constitutive responsiveness to extracellular dsRNA.

Distinct but partially overlapping signaling pathways mediate the response to extracellular vs. intracellular sources of dsRNA, by toll-like receptor 3 (TLR3) and retinoic acid-inducible gene-I/melanoma differentiated gene 5 (RIG-I/mda-5), respectively. Different cell types signal through these pathways to widely varying de grees. We previously observed that exposure to extracellular dsRNA, delivered by its addition to the culture medium, could induce the interferon (IFN)-stimulated gene 56 (ISG56) in human HT1080 fibrosarcoma cells, but not the HT1080-derived cell line, U3A, which lacks functional Stat1. In this study, we further investigated the nature of the dsRNA signaling defect in U3A cells. We show that a defect affecting basal TLR3 mRNA expression prevents U3A cells from responding to extracellular dsRNA. This defect does not impair dsRNA signaling in response to viral infection or transfected dsRNA. Although U3A cells are deficient in Stat1, we found that Stat1 was not required for basal TLR3 expression because other cell lines lacking Stat1 expressed TLR3. Moreover, restoration of Stat1 expression failed to restore TLR3 mRNA expression in U3A cells. However, treatment of Stat1-restored U3A cells with either IFN-beta or IFN-gamma induced TLR3 expression and restored responsiveness to extracellular dsRNA. Our results demonstrate that Stat1 is critical for IFN-induced, not basal, responsiveness to extracellular dsRNA.

ABCB5-Targeted Chemoresistance Reversal Inhibits Merkel Cell Carcinoma Growth.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer with profound but poorly understood resistance to chemotherapy, which poses a significant barrier to clinical MCC treatment. Here we show that ATP-binding cassette member B5 (ABCB5) confers resistance to standard-of-care MCC chemotherapeutic agents and provide proof-of-principle that ABCB5 blockade can inhibit human MCC tumor growth through sensitization to drug-induced cell cytotoxicity. ABCB5 expression was detected in both established MCC lines and clinical MCC specimens at levels significantly higher than those in normal skin. Carboplatin- and etoposide-resistant MCC cell lines exhibited increased expression of ABCB5, along with enhanced ABCB1 and ABCC3 transcript expression. ABCB5-expressing MCC cells in heterogeneous cancers preferentially survived treatment with carboplatin and etoposide in vitro and in human MCC xenograft-bearing mice in vivo. Moreover, patients with MCC also exhibited enhanced ABCB5 positivity after carboplatin- and etoposide-based chemotherapy, pointing to clinical significance of this chemoresistance mechanism. Importantly, ABCB5 blockade reversed MCC drug resistance and impaired tumor growth in xenotransplantation models in vivo. Our results establish ABCB5 as a chemoresistance mechanism in MCC and suggest utility of this molecular target for improved MCC therapy.

Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.

The skin of an adult human contains about 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice, but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human-engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All nonrecirculating resident memory T cells (TRM) expressed CD69, but most were CD4(+), CD103(-), and located in the dermis, in contrast to studies in mice. Both CD4(+) and CD8(+) CD103(+) TRM were enriched in the epidermis, had potent effector functions, and had a limited proliferative capacity compared to CD103(-) TRM. TRM of both types had more potent effector functions than recirculating T cells. We observed two distinct populations of recirculating T cells, CCR7(+)/L-selectin(+) central memory T cells (TCM) and CCR7(+)/L-selectin(-) T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions, and TMM were depleted more slowly from skin after alemtuzumab, suggesting that TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities.

TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.

Early diagnosis of cutaneous T cell lymphoma (CTCL) is difficult and takes on average 6 years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL, but the T cell receptor γ (TCRγ) polymerase chain reaction (PCR) analysis in current clinical use detects clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46 of 46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting that a niche of finite size may exist for benign T cells in skin. Last, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases, and provided insights into the cell of origin and location of malignant CTCL cells in skin.

Hybrid myxoinflammatory fibroblastic sarcoma/hemosiderotic fibrolipomatous tumor: report of a case providing further evidence for a pathogenetic link.

Myxoinflammatory fibroblastic sarcoma and hemosiderotic fibrolipomatous tumor are rare, slow-growing soft tissue tumors of the distal extremities with recurrent potential. Recent cytogenetic studies have shown a t(1;10)(p22;q24) or der(10)t(1;10) in combination with aberrations of chromosome 3 in a limited number of cases of both entities. Here we report a case of a 42-year-old female with a soft tissue tumor of the ankle showing hybrid morphologic features of myxoinflammatory fibroblastic sarcoma and hemosiderotic fibrolipomatous tumor, a der(10)t(1;10), and abnormalities of chromosome 3. This hybrid lesion provides further evidence for a close relationship between these 2 tumor types.

Human U2 snRNA genes exhibit a persistently open transcriptional state and promoter disassembly at metaphase.

In mammals, small multigene families generate spliceosomal U snRNAs that are nearly as abundant as rRNA. Using the tandemly repeated human U2 genes as a model, we show by footprinting with DNase I and permanganate that nearly all sequences between the enhancer-like distal sequence element and the initiation site are protected during interphase whereas the upstream half of the U2 snRNA coding region is exposed. We also show by chromatin immunoprecipitation that the SNAPc complex, which binds the TATA-like proximal sequence element, is removed at metaphase but remains bound under conditions that induce locus-specific metaphase fragility of the U2 genes, such as loss of CSB, BRCA1, or BRCA2 function, treatment with actinomycin D, or overexpression of the tetrameric p53 C terminus. We propose that the U2 snRNA promoter establishes a persistently open state to facilitate rapid reinitiation and perhaps also to bypass TFIIH-dependent promoter melting; this open state would then be disassembled to allow metaphase chromatin condensation.

Two tyrosine residues of Toll-like receptor 3 trigger different steps of NF-kappa B activation.

Innate immune response to viral infection is often triggered by Toll-like receptor 3 (TLR3)-mediated signaling by double-stranded (ds) RNA, which culminates in the activation of the transcription factor NF-kappaB and induction of NF-kappaB-driven genes. We demonstrated that dsRNA-induced phosphorylation of two specific tyrosine residues, 759 and 858, of TLR3 was necessary and sufficient for complete activation of the NF-kappaB pathway. When Tyr-759 of TLR3 was mutated, gene induction was inhibited, although NF-kappaB was partially activated. It was released from IkappaB and translocated to the nucleus but failed to bind to the kappaB site of the target A20 gene promoter. This defect could be attributed to incomplete phosphorylation of the RelA (p65) subunit of NF-kappaB, as revealed by two-dimensional gel analyses of p65, isolated from dsRNA-treated cells expressing either wild type TLR3 or the Tyr-759 --> Phe mutant TLR3. Thus, two phosphotyrosine residues of TLR3 activate two distinct pathways, one leading to NF-kappaB release and the other leading to its phosphorylation.

Analysis of genes induced by Sendai virus infection of mutant cell lines reveals essential roles of interferon regulatory factor 3, NF-kappaB, and interferon but not toll-like receptor 3.

Sendai virus (SeV) infection causes the transcriptional induction of many cellular genes that are also induced by interferon (IFN) or double-stranded RNA (dsRNA). We took advantage of various mutant cell lines to investigate the putative roles of the components of the IFN and dsRNA signaling pathways in the induction of those genes by SeV. Profiling the patterns of gene expression in SeV-infected cells demonstrated that Toll-like receptor 3, although essential for gene induction by dsRNA, was dispensable for gene induction by SeV. In contrast, Jak1, which mediates IFN signaling, was required for the induction of a small subset of genes by SeV. NF-kappaB and interferon regulatory factor 3 (IRF-3), the two major transcription factors activated by virus infection, were essential for the induction of two sets of genes by SeV. As expected, some of the IRF-3-dependent genes, such as ISG56, were more strongly induced by SeV in IRF-3-overexpressing cells. Surprisingly, in those cells, a number of NF-kappaB-dependent genes, such as the A20 gene, were induced poorly. Using a series of cell lines expressing increasing levels of IRF-3, we demonstrated that the degree of induction of A20 mRNA, upon SeV infection, was inversely proportional to the cellular level of IRF-3, whereas that of ISG56 mRNA was directly proportional. Thus, IRF-3 can suppress the expression of NF-kappaB-dependent genes in SeV-infected cells.

Natural mutations in a 2'-5' oligoadenylate synthetase transgene revealed residues essential for enzyme activity.

Unlike other RNA polymerases, 2'-5' oligoadenylate synthetases, a family of interferon-induced enzymes, catalyze the formation of 2'-5', not 3'-5', phosphodiester bonds. Moreover, to be active, these proteins require double-stranded RNA as a cofactor. We have been identifying the specific residues of these proteins that impart their novel properties. Here, we report the identity of three such residues that underwent natural mutations in a transgenic mouse line. When deliberately introduced into recombinant proteins, each of these mutations rendered the protein enzymatically inactive. In an effort to understand the roles of these residues in enzyme activity, new mutants carrying other residues in one of these three sites were generated. Detailed characterization of the properties of the mutant proteins revealed that Lys 404 is needed for proper binding of the acceptor substrate, Pro 500 provides structural flexibility to the protein, and Ser 471 is probably required for its proper folding. This study illustrates the power of using natural mutations in transgenes as guides for studying structure-function relationships of proteins.

Vid22p, a novel plasma membrane protein, is required for the fructose-1,6-bisphosphatase degradation pathway.

Fructose-1,6-bisphosphatase (FBPase), an important enzyme in the gluconeogenic pathway in Saccharomyces cerevisiae, is expressed when cells are grown in media containing a poor carbon source. Following glucose replenishment, FBPase is targeted from the cytosol to intermediate Vid (vacuole import and degradation) vesicles and then to the vacuole for degradation. Recently, several vid mutants that are unable to degrade FBPase in response to glucose were identified. Here, we present VID22, a novel gene involved in FBPase degradation. VID22 encodes a glycosylated integral membrane protein that localizes to the plasma membrane. Newly synthesized Vid22p was found in the cytoplasm and then targeted to the plasma membrane independent of the classical secretory pathway. A null mutation of VID22 failed to degrade FBPase following a glucose shift and accumulated FBPase in the cytosol. Furthermore, the majority of FBPase remained in a proteinase K sensitive compartment in the Deltavid22 mutant, implying that VID22 is involved in FBPase transport from the cytosol to Vid vesicles. By contrast, starvation-induced autophagy and peroxisome degradation were not impaired in the Deltavid22 mutant. This strain also exhibited the proper processing of carboxypeptidase Y and aminopeptidase I in the vacuole. Therefore, Vid22p appears to play a specific role in the FBPase trafficking pathway.