Search

While molecular testing of hematologic malignancies is now standard of care, there is variability in practice and testing capabilities between different academic laboratories, with common questions arising on how to best meet clinical expectations. A survey was sent to hematopathology subgroup members of the Genomics Organization for Academic Laboratories (GOAL) consortium to assess current and future practice and potentially establish a reference for peer institutions. Responses were received from eighteen academic tertiary-care laboratories regarding next-generation sequencing (NGS) panel design, sequencing protocols and metrics, assay characteristics, laboratory operations, case reimbursement, and development plans. Differences in NGS panel size, use, and gene content were reported. Gene content for myeloid processes was reported to be generally excellent while genes for lymphoid processes were less well covered. Turnaround time (TAT) for acute cases including acute myeloid leukemia was reported to range from 2-7 to 15-21 calendar days with different approaches to achieving rapid TAT described. To help guide NGS panel design and standardize gene content, consensus gene lists based on current and future NGS panels in development were generated. Most survey respondents expected molecular testing at academic laboratories to continue to be viable in the future with rapid TAT for acute cases likely to remain an important factor. Molecular testing reimbursement was reported to be a major concern. The results of this survey and subsequent discussions improve the shared understanding of differences in testing practices for hematologic malignancies between institutions and will help provide a more consistent level of patient care.

BackgroundExon 20 (ex20) in-frame insertions or duplications (ins/dup) in epidermal growth factor receptor (EGFR) and its analog erb-b2 receptor tyrosine kinase 2 (ERBB2) are each detected in 1.5% of non–small cell lung cancer (NSCLC). Unlike EGFR p.L858R or ex19 deletions, ex20 ins/dup is associated with de novo resistance to classic EGFR inhibitors, lack of response to immune checkpoint inhibitors, and poor prognosis. US Food and Drug Administration has approved mobocertinib and amivantamab for targeting tumors with this aberration, but the number of comprehensive studies on ex20 ins/dup NSCLC is limited. We identified 18 cases of NSCLCs with EGFR/ERBB2 ex20 ins/dup and correlated the findings with clinical and morphologic information including programed death-ligand 1 (PD-L1) expression.MethodsA total of 536 NSCLC cases tested at our institution between 2014 and 2023 were reviewed. A custom-designed 214-gene next-generation sequencing panel was used for detecting DNA variants, and the FusionPlex CTL panel (ArcherDx) was used for the detection of fusion transcripts from formalin-fixed, paraffin-embedded tissue. Immunohistochemistry (IHC)for PD-L1 was performed using 22C3 or E1L3N clones.ResultsNine EGFR and nine ERBB2 ex20 ins/dup variants were identified from an equal number of men and women, 14 were non- or light smokers, and 15 had stage IV disease. All 18 cases were adenocarcinomas. Seven of the 11 cases with available primary tumors had acinar predominant pattern, two had lepidic predominant pattern, and the remainder had papillary (one case) and mucinous (one case) patterns. Ex20 ins/dup variants were heterogenous in-frame one to four amino acids spanning A767–V774 in EGFR and Y772–P780 in ERBB2 and were clustered in the loop following the C-helix and α C-helix. Twelve cases (67%) had co-existing TP53 variants. Copy number variation in CDK4 amplification was identified in one case. No fusion or microsatellite instability was identified in any case. PD-L1 was positive in two cases, low positive in four cases, and negative in 11 cases.ConclusionsNSCLCs harboring EGFR/ERBB2 ex20 ins/dup are rare and tend to be acinar predominant, negative for PD-L1, more frequent in non- or light smokers, and mutually exclusive with other driver mutations in NSCLC. The correlation of different EGFR/ERBB2 ex20 ins/dup variants and co-existing mutations with response to targeted therapy and the possibility of developing resistant mutations after mobocertinib treatment warrants further investigation.

Multiple sclerosis (MS) is an autoimmune disease of the CNS in which the interaction between genetic and environmental factors plays an important role in disease pathogenesis. Although environmental factors account for 70% of disease risk, the exact environmental factors associated with MS are unknown. Recently, gut microbiota has emerged as a potential missing environmental factor linked with the pathobiology of MS. Yet, how genetic factors, such as HLA class II gene(s), interact with gut microbiota and influence MS is unclear. In the current study, we investigated whether HLA class II genes that regulate experimental autoimmune encephalomyelitis (EAE) and MS susceptibility also influence gut microbiota. Previously, we have shown that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to myelin proteolipid protein (91–110)–induced EAE, an animal model of MS, whereas AE-KO.HLA-DQ8 transgenic mice were resistant. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed higher disease prevalence and severity compared with HLA-DR3 mice. Gut microbiota analysis showed that HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 double transgenic mice microbiota are compositionally different from AE-KO mice. Within HLA class II transgenic mice, the microbiota of HLA-DQ8 mice were more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of DQ8 on an HLA-DR3 background increases disease severity, our data suggests that HLA-DQ8–specific microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota contribute to EAE susceptibility or resistance in a transgenic animal model of MS.

Objectives To identify therapeutic targets and correlate with clinical outcomes from mutation profiling of metastatic uveal melanoma (UM) using next-generation sequencing (NGS). Methods Melanoma cases that were tested using DNA-based NGS panels of 25 and/or 214 genes were evaluated retrospectively (263 cases) and identified 27 UM cases. BAP1 expression was examined by immunohistochemistry. Results Mutations in GNA11 (14) and GNAQ (12) were found in 96% (n = 27) of cases of UM, and most had coexisting BAP1 (17) or SF3B1 (4) mutations. Coexisting GNAQ/11-SF3B1 mutations correlated with a longer average time to first metastasis compared with GNAQ/11-BAP1 mutations (99.7 vs 38.5 months, P = .047). Three patients with BAP1 mutations received trametinib; two are still alive (15 months; 23 months), and one died (32 months). In non-UMs, only 4.2% (n = 236) had BAP1 and 3.8% had SF3B1 mutations; none had coexisting GNAQ/11 mutations. Conclusions Coexisting BAP1/SF3B1 and GNAQ/11 mutations were unique to UM. SF3B1 mutations were reported to be UM-specific in melanoma and associated with rare/no metastasis. The finding of mutated SF3B1 in 14.8% (n = 27) of UMs suggests its role should be further evaluated. The correlation of BAP1/SF3B1 mutation with survival also warrants investigation.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with unknown etiology. The interaction of both genetic and environmental factors plays an important role in MS pathogenesis. Recently, gut microbiota has emerged as a potential environmental factor in MS pathology due to its ability to modulate host immune response. Yet, it is unknown whether genetic factors, such as HLA class II gene(s), can influence gut microbiota which then can modulate MS through regulation of the host immune response. In the present study, we investigated the involvement of gut microbiota in HLA class II genes mediated susceptibility and resistance to MS/experimental autoimmune encephalomyelitis-EAE (an animal model of MS). Previously, we showed that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to proteolipid protein-PLP91–110 induced EAE, whereas AE-KO.HLA-DQ8 transgenic mice were resistant to PLP91–110. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed increased EAE severity compared with HLA-DR3 mice. Distinct microbiota in HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 transgenic mice compared to AE-KO mice suggested an important role of HLA class-II gene in shaping the gut microbiota composition. We also observed that gut microbiota of HLA-DQ8 mice was more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of HLA-DQ8 on an HLA-DR3 background increases disease severity, our data suggest that HLA-DQ8 restricted microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota may contribute to EAE susceptibility or resistance in a transgenic animal model of MS. The authors acknowledge funding from the National Multiple Sclerosis Society (RG 5138A1/1T), NIAID/NIH (1R01AI137075-01), a Carver Trust Medical Research Initiative Grant, and the University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH (P30 ES005605). SA was supported by the Emory Warner Fellowship, which provides medical students at the Carver College of Medicine the opportunity to take a full year out of their medical school curriculum to work in a laboratory in the University of Iowa Department of Pathology.

Trillions of microbes such as bacteria, fungi, and viruses exist in the healthy human gut microbiome. Although gut bacterial dysbiosis has been extensively studied in multiple sclerosis (MS), the significance of the fungal microbiome (mycobiome) is an understudied and neglected part of the intestinal microbiome in MS. The aim of this study was to characterize the gut mycobiome of patients with relapsing-remitting multiple sclerosis (RRMS), compare it to healthy controls, and examine its association with changes in the bacterial microbiome. We characterized and compared the mycobiome of 20 RRMS patients and 33 healthy controls (HC) using Internal Transcribed Spacer 2 (ITS2) and compared mycobiome interactions with the bacterial microbiome using 16S rRNA sequencing. Our results demonstrate an altered mycobiome in RRMS patients compared with HC. RRMS patients showed an increased abundance of Basidiomycota and decreased Ascomycota at the phylum level with an increased abundance of Candida and Epicoccum genera along with a decreased abundance of Saccharomyces compared to HC. We also observed an increased ITS2/16S ratio, altered fungal and bacterial associations, and altered fungal functional profiles in MS patients compared to HC.This study demonstrates that RRMS patients had a distinct mycobiome with associated changes in the bacterial microbiome compared to HC. There is an increased fungal to bacterial ratio as well as more diverse fungal-bacterial interactions in RRMS patients compared to HC. Our study is the first step towards future studies in delineating the mechanisms through which the fungal microbiome can influence MS disease.

BACKGROUND Molecular testing for epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) fusion is routinely performed in patients with stage IV lung adenocarcinoma to assess their eligibility for targeted therapy. Fine-needle aspiration (FNA)-derived material frequently is the only pathologic material available. The identification of genomic aberrations in thyroid nodules from FNA smears may help stratify cancer risk and spare patients from a second surgery. In the current study, the authors tested nucleic acid extracted from the cytology smears of lung and thyroid carcinomas for simultaneous detection of single-nucleotide variant, insertion/deletion, and gene fusion using an RNA-based next-generation sequencing assay. METHODS A total of 27 cases (17 lung and 10 thyroid carcinomas, the majority of which had known variants) were tested. Areas of interest were scrapped from stained smears using a scalpel. Total nucleic acid was extracted. Gene fusion and mutational analysis was performed using the Comprehensive Thyroid and Lung FusionPlex Assay. Data were analyzed using the analysis pipeline provided by the vendor. Eleven cases with available formalin-fixed, paraffin-embedded (FFPE) tissue were tested in parallel. RESULTS Gene fusions were detected in 6 cases; common single-nucleotide variants in EGFR, RAS, and BRAF in 14 cases; and in-frame deletions within EGFR in 3 cases. A concordance rate of 100% was observed between FNA and FFPE tissue. CONCLUSIONS Cytology preparations can be a reliable source for the detection of both DNA and RNA aberrations. The ability to simultaneously detect multiple types of genomic variants is crucial for patients with advanced cancer and maximizes the usefulness of cytology specimens. Cancer Cytopathol 2018;126:158-69. © 2018 American Cancer Society.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system, and among all the genes associated with MS, HLA class II genes have the highest relative risk. Although IL-17A is thought to be a major cytokine in the pathogenesis of MS, the exact role of IL-17A in the MS is still poorly understood. Specifically, it is unclear whether IL-17A is required for disease initiation or disease severity or in both. Therefore, in the present study, utilizing HLA-DR3 transgenic mice lacking IL-17A (HLA-DR3.IL-17A−/−), we investigated the importance of IL-17A in disease development and progression in experimental autoimmune encephalomnyelitis (EAE) an animal model of MS. We observed that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ regulatory T cells (Treg)s and developed milder EAE compared to IL-17 sufficient HLA-DR3 mice. Further characterization of mechanism identified a novel role of IL-17A in regulating the disease progression through modulation of immunoregulatory responses. We also identified an important role of gut microbiota in IL-17A dependent disease regulation specifically depletion of gut bacteria with ability to promote Treg population in IL-17A sufficient HLA-DR3 mice. Fecal transplantation of HLA.DR3 mice with HLA-DR3. IL17A−/− showed milder EAE and an increase in Treg cells in HLA-DR3 mice confirming a role of gut microbiota in shaping Treg repertoire and function. Additionally, we observed higher frequency of metabolic pathway involved with Treg promoting short chain fatty acid (SCFA) metabolism in HLA-DR3.IL17A−/− mice. Thus our study shows a novel role of IL-17A in immune homeostasis and inflammation by regulating levels of Tregs through modulation of gut microbiota.

Primary aneurysmal bone cyst (ABC) is a neoplastic process due to recurrent translocations involving the USP6 gene. By fluorescence in situ hybridization, up to 69% of primary ABCs harbored USP6 translocations; no USP6 translocation was found in secondary ABC or giant cell tumor of bone (GCT). GCT can recur locally, metastasize to the lungs in some cases, and rarely undergo malignant transformation. Differentiating primary ABC from its mimics is important for treatment and prognosis. We evaluated USP6 fusion and expression in 13 cases of primary and 1 case of secondary ABC, and 9 cases of GCT using nucleic acid extracted from formalin-fixed, paraffin-embedded tissue and a next generation sequencing (NGS)-based assay. USP6 fusions including 7 novel fusions and USP6 transcripts were identified in all 13 primary ABCs. Nine cases with strong evidence of fusions showed high levels of USP6 transcripts by reverse transcription-PCR (RT-PCR). The remaining four had no detectable USP6 expression by a first-round of RT-PCR but the presence of USP6 transcripts was identified by a second-round, nested PCR. The major fusions were confirmed by RT-PCR followed by Sanger sequencing. No USP6 fusion or transcript was detected in any of the GCTs or the case of secondary ABC by NGS or by two rounds of PCR. All USP6 translocations resulted in fusion of the entire USP6 coding sequence with promoters of the fusion gene leading to upregulation of USP6 transcription, which is likely the underlying mechanism for ABC oncogenesis. © 2016 Wiley Periodicals, Inc.

Solitary fibrous tumor (SFT) is a mesenchymal tumor of fibroblastic origin, which can affect any region of the body. 10-15% of SFTs metastasize and metastatic tumors are uniformly lethal with no effective therapies. The behavior of SFT is difficult to predict based on morphology. Recently, an intrachromosomal gene fusion between NAB2 and STAT6 was identified as the defining driving genetic event of SFT and different fusion types correlated with tumor histology and behavior. Due to the proximity of NAB2 and STAT6 on chromosome 12, this fusion may be missed by fluorescence in-situ hybridization. We evaluated 12 SFTs from 10 patients. All tumors showed strong nuclear staining for STAT6 by immunohistochemistry (IHC). The same formalin-fixed, paraffin-embedded blocks for IHC were used for gene fusion detection by a next-generation sequencing (NGS)-based assay. Targeted RNA fusion sequencing for gene fusions was performed using the Universal RNA Fusion Detection Kit, the Archer(™) FusionPlex(™) Sarcoma Panel and the Ion Torrent PGM, and data were analyzed using the Archer Analysis Pipeline 3.3. All tumors were positive for NAB2-STAT6 fusion. Six types of fusions were detected: NAB2ex4-STAT6ex2, NAB2ex2-STAT6ex5, NAB2ex6-STAT6ex16, NAB2ex6-STAT6ex17, NAB2ex3-STAT6ex18 and NAB2intron6-STAT6Ex17. The NGS findings were confirmed by RT-PCR followed by Sanger sequencing. No STAT6 fusion was detected in selected morphologic mimics of SFT. The assay also allows for detection of novel fusions and can detect NAB2-STAT6 fusions at a single-base resolution.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Despite IL-17A being linked to the immunopathogenesis of MS, the mechanism of IL-17A in disease development and maintenance is unclear. In the present study, we investigated the association of IL-17A and gut microbiota in disease development and severity, utilizing HLA-DR3 transgenic mice lacking IL-17A (IL-17A−/−). We found that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ Treg cells and develop milder EAE compared to IL-17A suffcient HLA-DR3 mice. Utilizing 16S metagenocmic sequencing and analysis of gut microbiota we observed that HLA-DR3.IL-17A−/− mice had an increased abundance of regulatory T cells promoting bacteria in the gut such as Lactobacillus and Clostridia. Fecal transplantation followed by co-housing of HLA.DR3 mice with HLA-DR3.IL17A−/− resulted in development of milder EAE and an increase in CD4+CD25+FoxP3+ Treg cells in HLA-DR3 mice. Further whole-genome shotgun metagenomic analysis showed modulation of pathogen-associated molecular patterns (PAMPs) and O-antigen pathways linked with the host innate immune responses in HLA-DR3.IL-17A−/− mice. Thus our study suggests that gut microbiota changes due to the absence of IL17A induce regulatory T cells and suppresses disease in an animal model of MS by modulating the host-bacterial dynamics via PRR-PAMPS interactions.

Multiple sclerosis (MS), an autoimmune disease of the central nervous system, has been shown to be influenced by gut bacteria. We and others have shown that MS patients have depletion of isoflavone-metabolizing bacteria compared to healthy individuals. We hypothesize that gut bacteria-mediated isoflavone metabolism induces an anti-inflammatory state and reduction/depletion of these gut bacteria might predispose to MS development and/or severity. To test this, we placed mice on a diet with or without isoflavones and induced EAE, an animal model of MS. Mice on an isoflavone diet developed milder disease compared to those on an isoflavone-free diet. This phenomenon was dependent on the presence of isoflavone-metabolizing gut bacteria as confirmed by antibiotic depletion and bacterial reconstitution. Mice on an isoflavone diet exhibited altered peripheral immune responses, CNS cellular infiltration, and CNS pathology post EAE induction compared to mice on an isoflavone-free diet. Additionally, the gut microbiome of mice on an isoflavone diet and an isoflavone-free diet closely resembles the gut microbiota of healthy individuals and MS patients, respectively. Thus our results suggest that dietary isoflavone metabolites produced with the help of gut bacteria can influence the disease through modulation of the immune system.

Molecular testing for epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) fusion is routinely performed in patients with stage IV lung adenocarcinoma to assess their eligibility for targeted therapy. Fine-needle aspiration (FNA)-derived material frequently is the only pathologic material available. The identification of genomic aberrations in thyroid nodules from FNA smears may help stratify cancer risk and spare patients from a second surgery. In the current study, the authors tested nucleic acid extracted from the cytology smears of lung and thyroid carcinomas for simultaneous detection of single-nucleotide variant, insertion/deletion, and gene fusion using an RNA-based next-generation sequencing assay.A total of 27 cases (17 lung and 10 thyroid carcinomas, the majority of which had known variants) were tested. Areas of interest were scrapped from stained smears using a scalpel. Total nucleic acid was extracted. Gene fusion and mutational analysis was performed using the Comprehensive Thyroid and Lung FusionPlex Assay. Data were analyzed using the analysis pipeline provided by the vendor. Eleven cases with available formalin-fixed, paraffin-embedded (FFPE) tissue were tested in parallel.Gene fusions were detected in 6 cases; common single-nucleotide variants in EGFR, RAS, and BRAF in 14 cases; and in-frame deletions within EGFR in 3 cases. A concordance rate of 100% was observed between FNA and FFPE tissue.Cytology preparations can be a reliable source for the detection of both DNA and RNA aberrations. The ability to simultaneously detect multiple types of genomic variants is crucial for patients with advanced cancer and maximizes the usefulness of cytology specimens. Cancer Cytopathol 2018;126:158-69. © 2018 American Cancer Society.

Primary aneurysmal bone cyst (ABC) is a neoplastic process due to recurrent translocations involving the USP6 gene. By fluorescence in situ hybridization, up to 69% of primary ABCs harbored USP6 translocations; no USP6 translocation was found in secondary ABC or giant cell tumor of bone (GCT). GCT can recur locally, metastasize to the lungs in some cases, and rarely undergo malignant transformation. Differentiating primary ABC from its mimics is important for treatment and prognosis. We evaluated USP6 fusion and expression in 13 cases of primary and 1 case of secondary ABC, and 9 cases of GCT using nucleic acid extracted from formalin-fixed, paraffin-embedded tissue and a next generation sequencing (NGS)-based assay. USP6 fusions including 7 novel fusions and USP6 transcripts were identified in all 13 primary ABCs. Nine cases with strong evidence of fusions showed high levels of USP6 transcripts by reverse transcription-PCR (RT-PCR). The remaining four had no detectable USP6 expression by a first-round of RT-PCR but the presence of USP6 transcripts was identified by a second-round, nested PCR. The major fusions were confirmed by RT-PCR followed by Sanger sequencing. No USP6 fusion or transcript was detected in any of the GCTs or the case of secondary ABC by NGS or by two rounds of PCR. All USP6 translocations resulted in fusion of the entire USP6 coding sequence with promoters of the fusion gene leading to upregulation of USP6 transcription, which is likely the underlying mechanism for ABC oncogenesis. © 2016 Wiley Periodicals, Inc.

Struma ovarii accounts for 5% of ovarian teratomas. Malignant transformation occurs in

Mutual regulation of expression between p53 and AR has been reported. To further investigate the role of p53 in the regulation of AR expression, an ARE-Luciferase vector was inserted into LNCaP and into LNCaP-sip53 transfectants, and AR activity was quantitatively estimated after treatment with proteasome inhibitors. LNCaP expresses a mutated form of AR. Therefore, to investigate whether p53 can modulate the expression of wild-type (wt) of AR, we transfected PC3-wtAR with a p53 vector together with ARE-Luc and showed that p53 expression decreased DHT-dependent activity of wtAR. Since proteasomes also participate in AR transcriptional activity, we investigated the role of p53 in proteasome-dependent inhibition of AR activity. More than 80% of AR activity was inhibited by 3 μM of lactacystin in LNCaP whereas no inhibition was noted in LN-sip53. We also found that lactacystin decreased AR-DNA binding 3-fold in LNCaP but no binding decrease was observed in LN-sip53. Taken together, our data show that the inhibitory effects of proteasome inhibitors are dependent on p53 status, at least in prostate cancer. Therefore, the role of p53 during treatment with proteasome inhibitors in different tumors should be further investigated.

Natalya V. Guseva 1 , Oskar W. Rokhlin 1 , Thomas B. Bair 2 , Rebecca B. Glover 1 and Michael B. Cohen 1 1 Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA 2 DNA Facility, University of Iowa, Iowa City, USA Received: February 13, 2012; Accepted: February 26, 2012; Published: February 29, 2012; Keywords: Androgen receptor; p53; FoxA1; prostate cancer Correspondence: Natalya V. Guseva, // // Abstract The androgen receptor (AR) is known to play a critical role in prostate cancer (PC). p53 likely also plays a role given that p53 mutations are commonly found in advanced PC, and loss of wild-type protein function contributes to the phenotype of castration-resistant prostate cancer (CRPC). Nevertheless, the extent of the contribution of p53 dysfunction to PC remains unclear. Here we analyze the effects of p53 inhibition in PC cells and show that it has significant consequences for both the interaction between AR, and chromatin and the proliferative capacity of these cells. Inhibition of p53 expression enabled LNCaP cells to proliferate independently of androgens. Moreover, it modified the genome-wide binding pattern of AR. ChIP-sequnce analyis (ChIP-seq) revealed that fewer AR-binding sites were present in the context of p53 inhibition, suggesting that wild-type p53 is required for stable binding of AR to certain chromatin regions. Further analysis revealed that a lower AR occupancy was accompanied by a reduction in FoxA1 binding at regulatory regions of AR-dependent genes. Our study also identifies a pool of genes that may be transcriptionally regulated by AR only in the absence of p53, and that may contribute to the CRPC phenotype. Overall, our results point to p53 playing an important role in regulating AR activity across the genome.

A key player in prostate cancer development and progression is the androgen receptor (AR). Tumor-associated lipogenesis can protect cancer cells from carcinogenic- and therapeutic-associated treatments. Increased synthesis of fatty acids and cholesterol is regulated by androgens through induction of several genes in androgen-responsive cancer cells. Acetyl-CoA-carboxylase-α (ACCA) is a key enzyme in the regulation of fatty acids synthesis. Here we show that AR binds in vivo to intron regions of human ACCA gene. We also show that the level of ACCA protein in LNCaP depends on AR expression and that DHT treatment increases ACCA expression and fatty acid synthesis. Inhibition of ACCA by TOFA (5-tetradecyl-oxy-2-furoic acid) decreases fatty acid synthesis and induces caspase activation and cell death in most PCa cell lines. Our data suggest that TOFA can kill cells via the mitochondrial pathway since we found cytochrome c release after TOFA treatment in androgen sensitive cell lines. The results also imply that the pro-apoptotic effect of TOFA may be mediated via a decrease of neuropilin-1(NRP1) and Mcl-1expression. We have previously reported that Mcl-1 is under AR regulation and plays an important role in resistance to drug-induced apoptosis in prostate cancer cells, and NRP1 is known to regulate Mcl-1 expression. Here, we show for the first time that NRP1 expression is under AR control. Taken together, our data suggest that TOFA is a potent cell death inducing agent in prostate cancer cells.

Natural killer (NK) cell-mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol-linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate-binding lectins, the role of glycosylation in ligand binding is unclear. Herein we show that m157 is expressed in multiple, differentially N-glycosylated isoforms in m157-transduced or MCMV-infected cells. We used site-directed mutagenesis to express single and combinatorial asparagine (N)-to-glutamine (Q) mutations at N178, N187, N213, and N267 in myeloid and fibroblast cell lines. Progressive loss of N-linked glycans leads to a significant reduction of total cellular m157 abundance, although all variably glycosylated m157 isoforms are expressed at the cell surface and retain the capacity to activate Ly49HB6 and Ly49I129 reporter cells and Ly49H+ NK cells. However, the complete lack of N-linked glycans on m157 destabilized the m157-Ly49H interaction and prevented physical transfer of m157 to Ly49H-expressing cells. Thus, glycosylation on m157 enhances expression and binding to Ly49H, factors that may impact the interaction between NK cells and MCMV in vivo where receptor-ligand interactions are more limiting.

The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.

The human prostatic carcinoma cell line DU145 has previously been found to be resistant to treatment with TNF-family ligands. However, TRAIL, TNF-alpha and anti-Fas antibodies (Ab) treatment in combination with the histone deacetylase inhibitor Trichostatin A (TSA) converted the phenotype of DU145 from resistant to sensitive. TSA induced 15% cell death but simultaneous treatment with TRAIL, TNF-alpha and anti-Fas Ab resulted in 55%, 70% and 40% cell death, respectively. Simultaneous treatment did not increase the level of TSA-induced histone acetylation, but induced the release of acetylated histones from chromatin into the cytosol. This release was caspase dependent since it was abrogated by Z-VAD-fmk. In addition, treatment with TSA induced caspase-9 activation and resulted in the release of cytochrome c and Smac/DIABLO from mitochondria. To further investigate the role of caspase-9 in TSA-mediated apoptosis we used two different approaches: (1) cells were pretreated with the caspase-9 inhibitor Z-LEHD-fmk, and (2) cells were transfected with a dominant-negative form of caspase-9. Both approaches gave similar results: cells became resistant to treatment with TSA. These data indicate that TSA mediates its effect via the mitochondrial pathway. This was confirmed by examining DU145 overexpressing Bcl-2. These transfectants were resistant to TSA treatment. Taken together, our data shows that only simultaneous treatment with TNF-family ligands and TSA in DU145 resulted in caspase activity sufficient to induce apoptosis. The combination of TSA and TNF-family ligands could potentially be the basis for the treatment of prostate cancer.

It has been suggested that some nuclear transcription factors may participate in the regulation of mitochondrial functions through transcriptional control of mitochondrial DNA. Very little is known about the response of transcription factors within mitochondria to the activation of death receptors. Recent publications indicate that nuclear factor-κB (NF-κB) is localized in mitochondria of mammalian cells. Because of the critical role of mitochondria in the execution of many apoptotic pathways, we suggest that NF-κB-dependent mechanisms operating at the level of mitochondria contribute to its role in regulating death receptor signaling. We have found NF-κB p65 and p50 subunits with DNA binding activity in the mitochondria of prostatic carcinoma cell lines. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) affects DNA binding activity of mitochondria-associated NF-κB but does not change the amount of p65 in mitochondria, which suggests activation of mitochondrial NF-κB without additional translocation of NF-κB subunits to mitochondria. We have also shown that TRAIL decreases mitochondrial genome encoded mRNA levels and inhibition of NF-κB prevents this decrease. TRAIL effects on mitochondrial NF-κB-DNA binding and mitochondrial genome encoded mRNA levels also depend on Bcl-2 overexpression. In addition, transcription factor activator protein-1 with DNA binding activity is also found in mitochondria of prostatic carcinoma cells and TRAIL treatment affects this binding. In summary, NF-κB is found in mitochondria of prostatic carcinoma cells, where it is thought to regulate mitochondria genome encoded mRNA levels in response to TRAIL treatment.

BACKGROUND Two main pathways of apoptosis in mammalian cells have been described: the death receptor pathway and the mitochondrial pathway. Two different cell types have been identified for Fas-mediated apoptosis, each using almost exclusively one of two different signaling pathways. Human prostatic carcinoma cell line, PC3 is sensitive to Fas-mediated apoptosis, but relation of receptor and mitochondrial pathways is not clear. METHODS Cell viability was estimated by calcein assay. Apoptosis was determined by preparation of DNA ladder. Expression of Fas-associated death domain-dominant negative (FADD-DN) and Bcl-2, activation of caspases, PARP, DFF45, Bid cleavage, and cytochrome c release were assessed using Western blotting techniques. [35S] Methionine-labeled caspase-3 was transcribed in vitro and translated using the TNT kit (Promega). A vector containing caspase-3 was prepared by the ligation of EcoR I/BamHI flanked PCR fragment of full size caspase-3 cDNA into pBlusckript II SK(+/−) (Stratagen). RESULTS Overexpression of both FADD-DN and Bcl-2 genes prevent Fas-mediated apoptosis in PC3. As predicted, overexpression of FADD-DN prevented activation of caspase-8 and Bid cleavage and attenuated the release of cytochrome c and activation of caspases -2, -7, and -9. Bcl-2 overexpression did not affect caspase-8 activation and cleavage of Bid but blocked the release of cytochrome c and activation of mitochondria localized caspases -2, -7, and–9. Overexpression of FADD-DN and Bcl-2 affected the activation of caspase-3 and PARP cleavage differently: FADD-DN attenuated the activation of caspase-3 and PARP cleavage whereas Bcl-2 overexpression prevented caspase-3 activation and completely blocked cleavage of PARP. CONCLUSIONS These data suggest that activation of caspase-8 is necessary but not sufficient to complete Fas-mediated apoptosis in PC3 cells without activation of the mitochondrial pathway. In addition, caspase-3 activation after Fas-receptor ligation involves two steps and is dependent on mitochondrial activation. Prostate 51: 231–240, 2002. © 2002 Wiley-Liss, Inc.

It has been suggested that the downregulation of AR expression should be considered the principal strategy for the treatment of hormone-refractory prostate cancer. We have previously shown that inhibition of AR induced PI3K-independent activation of Akt that was mediated by CaMKII. In this study, we found that the CaMKII inhibitor KN-93 has a broader effect on apoptosis than just inhibition of CaMKII: first, KN-93 inhibits AR activity and induces cell death in PCa cells after androgen deprivation when many other drugs fail to kill prostate cancer cells; second, KN-93 inhibits expression of the anti-apoptotic protein Mcl-1 and induces expression of the pro-apoptotic protein PUMA; third, KN-93-mediated cell death is p53-independent; and fourth, KN-93 induces the generation of ROS. The ROS induction allows KN-93 to circumvent the activation of Akt, which occurs in prostate cancer cells under androgen deprivation, since Akt could not inhibit ROS-mediated apoptosis. KN-93 also synergistically induces cell death in combination with low doses of doxorubicin and converts the phenotype of prostate cancer cells from TRAIL-resistant to -sensitive. These data suggest that KN-93 could be used for novel therapeutic approaches when hormonal therapy has failed.

Background: AML is a clinically and genetically heterogeneous clonal disorder. Approximately 50% are characterized by recurrent clonal chromosome aberrations which have contributed to the classification of disease and are recognized as important prognostic factors. AML patients who lack these recurrent structural abnormalities have been grouped as “intermediate cytogenetic risk” and are being further subcategorized by the sequence alterations that are being identified. A more complete understanding of the genetic changes that are relevant to the pathogenesis of AML will improve the classification of risk and ultimately better selection of therapy. Our hypothesis is that mutational profiling and analysis of patient outcomes will help better define the risk subgroups of patients and predict prognosis in patients with AML. Methods: Archived DNA from 37 patients with various AML diagnoses were obtained with IRB approval (IRB#201502763). A panel of 30 commonly mutated genes in AML were designed using the Ion Ampliseq Designer (Ion Torrent) and targeted for “hotspot” or complete coding sequence (CDS). Genomic libraries were prepared using Ion AmpliSeq™2.0 (ThermoFisher) technology. Library clonal amplification was performed by emulsion PCR using Ion PGM™ Template OT2 Reagents Kit200. Simultaneous sequencing of clonally amplified DNA targets was performed by Ion-Torrent Personalized Genome Machine (IT-PGM) using 318v2 chip. Genomic variants were identified by variant caller3.4 and further filtered and analysed through a custom-made clinically validated pipeline. Novel mutations were confirmed by either single nucleotide primer extension or Sanger sequencing. Results: The assay successfully identified previously identified point mutations in FLT-3 and CEBPA genes. Additional variants were identified in 81% (30/37) of specimens; no mutations were found in 19% (7/37) specimens. The genes most frequently mutated were DNMT3A (23%), TET2 (17%), IDH2 (14%) and TP53 (14%). U2AF1 (11%) and RUNX1 (8%) were exclusively mutated in specimens with non-recurrent cytogenetic abnormalities. The identified variants will be used to risk stratify patients based on clinical outcome. Conclusion: This targeted multi-gene panel successfully identified several variants along with previously known mutations in archived specimens. Citation Format: Anup Tilak, Ramakrishna Sompallae, Aaron Stence, Natalya Guseva, Aaron Bossler. Development of a targeted panel for acute myeloid leukemia (AML) by using next-generation sequencing (NGS). [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 01.

The majority of breast cancer cell lines are resistant to tumor necrosis factor -related apoptosis inducing ligand (TRAIL) induced apoptosis. TRAIL and Fas receptor death-inducing signaling complex (DISCs) formation are similar and involve ligand-dependent recruitment of FADD and caspase-8. We have found that the breast carcinoma cell line T47D is an unusual example of selective sensitivity to anti-Fas mAb treatment but resistant to TRAIL. Therefore, a detailed comparison of these two signaling pathways in one cell line should provide insight into the mechanism of TRAIL resistance. We observed that only anti-Fas mAb induces caspase activation and cell death in T47D. Further, FADD and caspase-8 interact with both TRAIL-R1 and TRAIL-R2, and that the amount of caspase-8 recruited by Fas-, TRAIL-R1 and TRAIL-R2 are the same. cFLIP(S) and cFLIP(R )isoforms block death receptor-induced apoptosis by inhibiting caspase-8 activation at the DISC; the role of cFLIP(L )at the DISC is still controversial. It has been suggested that the presence of the cleaved form of FLIP(L)-p43 at the DISC prevents caspase-8 cleavage. We found that both TRAIL and anti-Fas mAb-induced DISCs contain the cleaved form of p43 cFLIP(L) and its amount at the Fas DISC was higher compared to the TRAIL DISC. We also found that inhibition of cFLIP(L) expression in T47D cells decreased Fas-mediated caspase-8 activation and activation of effector caspases. We propose that in T47D p43 cFLIP(L) in the Fas-DISC may promote caspase-8 activation. The mechanism by which different amounts of p43cFLIP(L) regulates caspase-8 activation remains to be investigated.

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)–positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A–induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A–induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A–induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21. (Mol Cancer Res 2006;4(2):113–23)

Background: Invasive micropapillary carcinoma (IMPC) of the breast is an uncommon disease subtype that is generally considered aggressive. A variety of carcinomas can exhibit micropapillary histology including lung, bladder, and ovary. Immunohistochemical (IHC) and genomic studies at these other organ sites have identified potential therapeutic targets including BRAF mutations. The purpose of this study was to characterize the IHC and molecular profile of a cohort of IMPC that could suggest potentially useful targeted therapies. Design: Eight tumor blocks (from seven patients) of IMPC were identified from the archives at the University of Iowa Hospitals and Clinics from 1997-2012. IHC stains for ER, PR, HER2, PTEN (Dako; clone 6H2.1, 1:100) and NM23H1 (Santa Cruz Biotechnology, clone: Nm301, 1:50) were performed. ER, PR, and HER2 were score according to ASCO/CAP guidelines. PTEN and NM23H1 were scored using a 4-tier system: P: strong homogenous; H: heterogeneous (positive with negative foci); R: reduced in more than 50%; and L: lost in all or majority. Genomic DNA was isolated from the formalin fixed paraffin embedded tumor samples used for IHC. BRAF V600 mutational analysis was analyzed using a laboratory-developed primer extension assay. Massive parallel sequencing (next generation sequencing, NGS) was performed using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA). Results: The patients were 50-83 years of age. Six had Elston-Ellis Grade II tumors and one was Elston-Ellis grade I. Four patients had axillary lymph node metastases. All cases examined showed strong and diffuse estrogen and progesterone receptor expression with no over-expression of HER2. Complete PTEN loss was observed in 4 cases including 3 with lymph node positive disease, three of these four cases had concurrent reduced expression of NM23H1. No mutations in exon 15 of the BRAF gene were detected. By NGS, one sample showed a KITc.A1621C (pM541) polymorphism, three had no mutations, and sequencing from the other three was unsatisfactory. Molecular and IHC Profile of Seven Patients with IMPC of the BreastHistologic GradeAxillary Lymph Node MetastasisER/PRHER2PTENNM23H1NGS2NoPositive/PositiveNegativePPUnsatisfactory1NoPositive/PositiveNegativeLRNegative2NoPositive/PositiveNegativeHPIntronic2YesPositive/PositiveNegativeHPUnsatisfactory2YesPositive/PositiveNegativeLRKIT c.A1621C (pM541)2YesPositive/PositiveNegativeLPNegative2YesPositive/PositiveNegativeLRUnsatisfactory Conclusions: Loss of PTEN expression was observed in 57% of cases (4/7) and was often associated with lymph node metastasis. A direct correlation between PTEN and NM23H1 expression was observed in six cases. No BRAF or other mutations were detected. PTEN was included in the NGS panel. Reduced expression observed in our study is most likely due to posttranscriptional modifications. Concurrent reduced expression of the metastatic suppressor NM23H1 may play a role in the clinical behavior of IMPC. The high frequency of PTEN loss may have implications for targeted therapy towards the PI3K pathway in this subgroup of patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-14.

It has been suggested that some nuclear transcription factors may participate in the regulation of mitochondrial functions through transcriptional control of mitochondrial DNA. Very little is known about the response of transcription factors within mitochondria to the activation of death receptors. Recent publications indicate that nuclear factor-kappaB (NF-kappaB) is localized in mitochondria of mammalian cells. Because of the critical role of mitochondria in the execution of many apoptotic pathways, we suggest that NF-kappaB-dependent mechanisms operating at the level of mitochondria contribute to its role in regulating death receptor signaling. We have found NF-kappaB p65 and p50 subunits with DNA binding activity in the mitochondria of prostatic carcinoma cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) affects DNA binding activity of mitochondria-associated NF-kappaB but does not change the amount of p65 in mitochondria, which suggests activation of mitochondrial NF-kappaB without additional translocation of NF-kappaB subunits to mitochondria. We have also shown that TRAIL decreases mitochondrial genome encoded mRNA levels and inhibition of NF-kappaB prevents this decrease. TRAIL effects on mitochondrial NF-kappaB-DNA binding and mitochondrial genome encoded mRNA levels also depend on Bcl-2 overexpression. In addition, transcription factor activator protein-1 with DNA binding activity is also found in mitochondria of prostatic carcinoma cells and TRAIL treatment affects this binding. In summary, NF-kappaB is found in mitochondria of prostatic carcinoma cells, where it is thought to regulate mitochondria genome encoded mRNA levels in response to TRAIL treatment.

TPCK is widely used as an inhibitor of chymotrypsin-like proteases but has recently been identified as an inhibitor of the PDK1/Akt pathway. In this study, we show that TPCK inhibits TRAIL-induced caspase activity but potentiates wortmannin-dependent caspase activity in prostatic carcinoma cell lines. The inhibitory activity of TPCK was found to be death ligand-specific since TPCK inhibits TRAIL-mediated caspase activity but does not affect Fas-induced caspase activity. Our data also show that impaired TRAIL-DISC formation in the presence of TPCK is responsible for caspase inhibition. Further, TPCK induces p53 expression and inhibits the PDK1/Akt pathway resulting in BAD dephosphorylation, and the release of cytochrome c and Smac/DIABLO from mitochondria. TPCK also selectively decreases the levels of androgen receptor and caspase-2 whereas it does not change the levels of other proteins (caspases-3, -7, -8, -9; heat shock proteins 27, 70, 90). Finally, TPCK-induced degradation of caspase-2 is protected by Bcl-2 overexpression, apparently by an adapter protein since direct interaction between caspase-2 and Bcl-2 was not detected. Together, these features suggest that TPCK could be used as a therapeutic agent for treatment of those tumor cells that are resistant to ligand-induced treatment because of aberrant signaling pathways downstream of the DISC.

Prostate cancer mortality results from metastasis and is often coupled with progression from androgen-dependent to androgen-independent growth. Unfortunately, no effective treatment for metastatic prostate cancer increasing patient survival is available. The absence of effective therapies reflects in part a lack of knowledge about the molecular mechanisms involved in the development and progression of this disease. Apoptosis, or programmed cell death, is a cell suicide mechanism that enables multicellular organisms to regulate cell number in tissues. Inhibition of apoptosis appears to be a critical pathophysiological factor contributing to the development and progression of prostate cancer. Understanding the mechanism(s) of apoptosis inhibition may be the basis for developing more effective therapeutic approaches. Our understanding of apoptosis in prostate cancer is relatively limited when compared to other malignancies, in particular, hematopoietic tumors. Thus, a clear need for a better understanding of apoptosis in this malignancy remains. In this review we have focused on what is known about apoptosis in prostate cancer and, more specifically, the receptor/ligand-mediated pathways of apoptosis as potential therapeutic targets.

Here we show that LNCaP, which is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, becomes sensitive to TRAIL after overexpression of full-length, wild-type BAD (BAD WT). TRAIL induces caspase-dependent cleavage of BAD WT that results in generation of a M(r) 15,000 protein. LNCaP stably expressing truncated BAD (tBAD) and cells expressing mutated BAD at the caspase cleavage site were less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Cytochrome c and Smac/DIABLO release from mitochondria into cytosol was found after TRAIL treatment only in cells overexpressing BAD WT. Furthermore, differences in phosphorylation of serine residues for BAD WT and tBAD were identified. BAD WT was phosphorylated at positions S136 and S155, whereas tBAD was phosphorylated at positions S112, S136, and S155. LNCaP stably expressing BAD mutated at serine 112 to alanine was less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Lastly, recombinant BAD cleaved by caspase-3 is a more potent inducer of cytochrome c and Smac/DIABLO release than BAD WT. In summary, BAD-mediated sensitivity of LNCaP to TRAIL depends on the phosphorylation status of BAD WT and tBAD.

Bisindolylmaleimides (Bis) were originally described as protein kinase C inhibitors. Several studies have shown that Bis potentiate tumor necrosis factor (TNF) receptor family-mediated apoptosis in lymphoid and dendritic cells, but the inhibition of protein kinase C cannot account for these effects (Zhou, T., Song, L., Yang, P., Wang, Z., Lui, D., and Jope, R. S. (1999) Nat. Med. 5, 42-48). We investigated the effect of four Bis derivatives (I, II, VIII, and IX) in human prostatic carcinoma cell lines and found that Bis IX was the most potent inducer of apoptosis under simultaneous treatment with TNF-alpha, agonistic anti-Fas monoclonal antibody, and TNF-related apoptosis-inducing ligand (TRAIL). Bis IX synergistically induced caspase activity in combination with apoptosis-inducing ligands and converted the phenotype of cell lines from apoptosis-resistant to -sensitive. Bis IX induced p53 accumulation in LNCaP (lymph node carcinoma of prostate), which expresses wild-type p53 that was not accompanied by the induction of p53-responsive genes, p21/WAF1, and Mdm2. Moreover, the induction of p21/WAF1 and Mdm2 by doxorubicin was abrogated by simultaneous treatment with Bis IX. These effects apparently result from general inhibition of transcription by Bis IX. We have shown by Northern blot analysis that the transcription activity of the hygromycin gene after transient transfection of pcDNA3.1-Hygro plasmid in 293 and HeLa cells was inhibited by Bis IX in a dose-dependent manner. Moreover, DNA binding activity of Bis IX was prevented by actinomycin D, suggesting that actinomycin D and Bis IX have similar mechanisms of interaction with DNA. In addition, we found that actinomycin D and Bis IX induced caspase activity to the same extent during TRAIL-mediated apoptosis. In summary, these results suggest that Bis IX potentiates TNF receptor family-mediated cell death in part as an inhibitor of transcription.

BACKGROUND The differential sensitivity of tumor cells to TRAIL-induced apoptosis may be mediated by different intracellular inhibitors of apoptosis, and only a few reports have described the pathway(s) that are activated in response to TRAIL in prostate cells. METHODS LNCaP was transfected with a dominant-negative form of FADD (FADD-DN) and cells were selected in the presence of hygromycin. Cell viability was estimated by calcein assay. Apoptosis was estimated by caspase activation using both fluorogenic substrastes and Western blot analysis of activated caspases. To detect cytochrome c release, mitochondria-free cytosol was prepared and Western blot analysis was performed. RESULTS LNCaP is resistant to TRAIL but TRAIL transiently induces DEVDase activity and activation of caspase-8; caspase-2, -3, -7, and -9 were not activated. Wortmannin, an inhibitor of the PI3K/Akt pathway, converted the phenotype of LNCaP from TRAIL-resistant to -sensitive. In the presence of wortmannin TRAIL induced activation of caspase-2, -3, -7, -8, and -9, as well as dissipation of mitochondrial transmembrane potential and release of cyto-chrome c from mitochondria into the cytosol. In addition, combined TRAIL and wortmannin treatment resulted in cleavage of several proteins: PARP, Akt, p21/WAF1, and MDM2 as well as dephosphorylation of Akt. The proteolysis of p21/WAFI and Akt, which are known survival factors, presumably amplify the apoptotic cascade in LNCaP. Transfection of FADD-DN in LNCaP resulted in inhibition of caspase activation as well as in resistance to combined treatment with TRAIL and wortmannin. CONCLUSIONS These results suggest that caspase-8 activation is necessary but not sufficient for TRAIL-mediated apoptosis and is presumably blocked downstream of caspase-8 by the PI3K/Akt pathway. Prostate 52: 1–11, 2002. © 2002 Wiley-Liss, Inc.

Alexandra Thomas,1 Ryan W Askeland,2 Natalya V Guseva,2 Ramakrishna Sompallae,2,3 Deqin Ma2 1Department of Internal Medicine, 2Department of Pathology, 3Bioinformatics Division, Iowa Institute of Human Genetics, University of Iowa Hospitals and Clinics, Iowa City, IA, USABackground: In this study, molecular and immunohistochemical profiling of invasive micropapillary carcinoma of the breast was used to identify potentially useful markers for targeted therapies with a focus on BRAF V600E mutation.Methods: Formalin-fixed, paraffin-embedded tumor blocks from seven patients were identified from the archives at our institution and tumor registry from 1997 to 2012. Massively parallel (Next-generation) sequencing was performed using the Ion AmpliSeq™ Cancer Hotspot Panel version 2 (Life Technologies, Carlsbad, CA, USA). Mutation analysis for BRAF V600E was performed using a single nucleotide primer extension assay. Immunohistochemistry studies for estrogen receptor (ER), progesterone receptor (PR), Her2/Neu, phosphatase and tensin homolog (PTEN), and non-metastatic protein 23 homologue 1 (NM23H1) were performed using the same tumor blocks. Staining for ER, PR, and Her2/Neu was scored according to American Society of Clinical Oncology/College of American Pathologists guidelines, and a four-tier system, ie, strong homogenous, heterogeneous, positive with negative foci, reduced in more than 50%, and lost in all or majority was used for PTEN and NM23H1 staining.Results: No pathogenic mutations were identified in the tumors by next-generation sequencing. The lack of BRAF V600E mutation was confirmed by single nucleotide primer extension assay. All tumors were positive for ER and PR, and showed no overexpression of Her2/Neu. Loss of or reduced PTEN expression was observed in six of seven cases and was associated with lymph node metastasis. Reduced NM23H1 expression was observed in three of seven cases, all of which had concurrent PTEN loss.Conclusion: No somatic mutation was identified consistent with the reported lack of driver mutations in this tumor. The frequent loss of PTEN in invasive micropapillary carcinoma may have implications for targeted therapy towards the phosphatidylinositol-4,5-bisphosphate 3-kinase pathway in this subgroup of patients.Keywords: invasive micropapillary carcinoma, next-generation sequencing, immunohistochemistry

The murine cytomegalovirus-encoded protein m157 is a cognate ligand for both inhibitory and activating receptors expressed by natural killer cells. Additionally, m157 is expressed on the surface of infected cells by a glycophosphatidylinositol (GPI) anchor. Although endogenous GPI-anchored proteins are known to be ligands for the NK cell receptor, NKG2D, the contribution of the GPI anchor for viral m157 ligand function is unknown. To determine whether the GPI anchor for m157 is dispensable for m157 function, we generated m157 variants expressed as transmembrane fusion proteins and tested cells expressing transmembrane m157 for the capacity to activate cognate Ly49 receptors. We found that the GPI anchor is required for high-level cell surface expression of m157, and that the transmembrane m157 ligand retains the capacity to activate reporter cells and NK cells expressing Ly49H, as well as Ly49I(129) reporter cells, but with reduced potency. Importantly, target cells expressing the transmembrane form of m157 were killed less efficiently and failed to mediate Ly49H receptor downregulation on fresh NK cells compared to targets expressing GPI-anchored m157. Taken together, these results show that the GPI anchor for m157 facilitates robust cell surface expression, and that NK cells are sensitive to the altered cell surface expression of this potent viral evasin.

The MCMV-encoded glycoprotein, m157, is recognized by the activating Ly49HC57Bl/6 and inhibitory Ly49I129S lectin-like NK cell receptors. We and others have shown that Ly49H is tolerant to multiple amino acid changes in m157 with the exception of K161, which is predicted to anchor a unique N-terminal α0-helix to the main m157 chain. We undertook site-directed mutagenesis to address the contributions of N-linked glycosylation and the GPI anchor of m157 to Ly49H and Ly49I recognition. Variants of m157 containing single or multiple N-to-Q substitutions at positions N178, N187, N213, and N267, or fusions between the m157 extracellular domain and the transmembrane + cytoplasmic domains of DAP12 or CD4, were expressed in myeloid or fibroblast cell lines. We find that 1) only fully deglycosylated m157 has reduced capacity to activate Ly49H or chimeric Ly49HI reporter cells; 2) m157-CD4 transmembrane versions of m157 fully activate Ly49H and chimeric Ly49HI reporter lines; and 3) Ly49H+ NK cells are activated by all N-glycosylation and transmembrane variants of m157. Thus, neither N-glycosylation nor GPI anchoring of m157 is required for functional trans interactions with lectin-like Ly49 receptors.