Candidate For Targeted Therapy Research Articles

SYVN1 Promotes STAT3 Protein Ubiquitination and Exerts Antiangiogenesis Effects in Retinopathy of Prematurity Development.

SYVN1, a gene involved in endoplasmic reticulum-associated degradation, has been found to exert a protective effect by inhibiting inflammation in retinopathy. This study aimed to clarify whether SYVN1 is involved in the pathogenesis of retinopathy of prematurity (ROP) and its potential as a candidate for target therapy. Human retinal microvascular endothelial cells (hRMECs) and a mouse model of oxygen-induced retinopathy (OIR) were used to reveal the retinopathy development-associated protein expression and molecular mechanism. An adenovirus overexpressing SYVN1 or vehicle control was injected intravitreally at postnatal day 12 (P12), and the neovascular lesions were evaluated in retinal flatmounts with immunofluorescence staining, and hematoxylin and eosin staining at P17. Visual function was assessed by using electroretinogram (ERG). Endogenous SYVN1 expression dramatically decreased in hRMECs under hypoxia and in ROP mouse retinas. SYVN1 regulated the signal transducer and activator of transcription 3 (STAT3)/vascular endothelial growth factor (VEGF) axis. SYVN1 overexpression promoted ubiquitination and degradation of STAT3, decreased the levels of phospho-STAT3, secretion of VEGF, and formation of neovascularization in hRMECs, which could be rescued by STAT3 activator treatment. In addition, SYVN1 overexpression prevented neovascularization and extended physiologic retinal vascular development in the retinal tissues of OIR mice without affecting retinal function. SYVN1 has a protective effect against OIR, and the molecular mechanisms are partly through SYVN1-mediated ubiquitination of STAT3 and the subsequent downregulation of VEGF. These findings strongly support our assumption that SYVN1 confers ROP resistance and may be a potentially novel pharmaceutical target against proliferative retinopathy.

R-spondin-3 is an oncogenic driver of poorly differentiated invasive breast cancer.

R-spondins (RSPOs) are influential signaling molecules that promote the Wnt/β-catenin pathway and self-renewal of stem cells. Currently, RSPOs are emerging as clinically relevant oncogenes, being linked to cancer development in multiple organs. Although this has instigated the rapid development and testing of therapeutic antibodies targeting RSPOs, functional evidence that RSPO causally drives cancer has focused primarily on the intestinal tract. Here, we assess the oncogenic capacity of RSPO in breast cancer in a direct fashion by generating and characterizing a novel mouse model with conditional Rspo3 expression in the mammary gland. We also address the prevalence of RSPO gene alterations in breast cancer patients. We found that a quarter of breast cancer patients harbor RSPO2/RSPO3 copy number amplifications, which are associated with lack of steroid hormone receptor expression and reduced patient survival. Foremost, we demonstrate the causal oncogenic capacity of RSPO3 in the breast, as conditional Rspo3 overexpression consistently drives the development of mammary adenocarcinomas in our novel Rspo3 breast cancer model. RSPO3-driven mammary tumors typically show poor differentiation, areas of epithelial-to-mesenchymal transition, and metastatic potential. Given the reported interplay in the Wnt/β-catenin pathway, we comparatively analyzed RSPO3-driven mouse mammary tumors versus classical WNT1-driven analogues. This revealed that RSPO3-driven tumors are distinct, as the poorly differentiated tumor morphology and metastatic potential were observed in RSPO3-driven tumorigenesis exclusively, further substantiated by differentiating gene expression profiles. Co-expression of Rspo3 and Wnt1 transduced mammary tumors with a mixed phenotype harboring morphological features characteristic of both transgenes. In summary, we report that a quarter of breast cancer patients harbor RSPO2/RSPO3 copy number gains, and these patients have a worse prognosis, whilst providing in vivo evidence that RSPO3 drives poorly differentiated invasive breast cancer in mice. Herewith, we establish RSPO3 as a driver of breast cancer with clinical relevance, proposing RSPO3 as a novel candidate target for therapy in breast cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Plasma Cytokines Profile in Subjects with Alzheimer's Disease: Interleukin 1 Alpha as a Candidate for Target Therapy.

Background:Testicular function depends on temperature, and it has been shown that scrotal hyperthermia causes a sharp decrease in sperm parameters due to oxidative stress. In recent years, the use of natural materials from the plant and nanoparticles has attracted much attention. Therefore, the present study aimed to investigate the effect of curcumin and Fe2O3 nanoparticles on sperm parameters in rats.Materials and Methods:After preparing the rats, they were placed in a hot water bath at 43°C for 30 minutes for six consecutive days. The 48 rats were then divided into eight groups. A concentration of 0.03 mg/kg body weight magnetic Fe2O3 nanoparticles and curcumin at the concentration of 0.02 mg/kg body weight were used. After killing animals, the semen parameters such as viability, concentration, motility, and morphology of sperm were studied.Results:Significant differences were observed in all groups of rats in terms of semen parameters (P<0.001). The results showed a positive effect of curcumin on improving semen parameters in scrotal hyperthermia rats and a negative and toxic effect of Fe2O3 magnetic nanoparticles. However, significant improvement in sperm parameters was observed when Fe2O3 magnetic nanoparticles were given to rats along with curcumin. Conclusion:Curcumin has a positive and significant effect on improving sperm parameters in scrotal hyperthermia conditions. Fe2O3 magnetic nanoparticles, if co-administered with curcumin, can significantly improve sperm parameters. In this regard, green synthesis of nanoparticles and concomitant administration of antioxidants such as curcumin in scrotal hyperthermia conditions is recommended.

Plasma Cytokines Profile in Subjects with Alzheimer's Disease: Interleukin 1 Alpha as a Candidate for Target Therapy.

Background:Alzheimer’s disease (AD) is the main cause of the neurodegenerative disorder, which is not detected unless the cognitive deficits are manifested. An early prediagnostic specific biomarker preferably detectable in plasma and hence non-invasive is highly sought-after. Various hypotheses refer to AD, with amyloid-beta (Aβ) being the most studied hypothesis and inflammation being the most recent theory wherein pro-and anti-inflammatory cytokines are the main culprits.Materials and Methods:In this study, the cognitive performance of AD patients (n=39) was assessed using mini-mental state examination (MMSE), AD assessment scale-cognitive subscale (ADAS-cog), and clinical dementia rating (CDR). Their neuropsychiatric symptoms were evaluated through neuropsychiatric inventory–questionnaire (NPI-Q). Moreover, plasma levels of routine biochemical markers, pro-/anti-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1 α (IL-1α), IL-1β, IL-2, IL-4, IL-6, IL-8, IL-12p70, IL-10, Interferon-gamma, chemokines, including prostaglandin E2 (PGE-2), monocyte chemoattractant protein-1, interferon gamma-induced protein 10, Aβ peptide species (42, 40) and Transthyretin (TTR) were measured.Results:Our results revealed that Aβ 42/40 ratio and TTR were correlated (r=0.367, P=0.037). IL-1α was directly correlated with ADAS-cog (r=0.386, P=0.017) and Aβ 40 (r=0.379, P=0.019), but was inversely correlated with IL-4 (r=-0.406, P=0.011). Negative correlations were found between MMSE and PGE2 (r=-0.405, P=0.012) and TNF-α/ IL-10 ratio (r=-0.35, P=0.037). CDR was positively correlated with both PGE2 (r=0.358, P=0.027) and TNF-α (r=0.416, P=0.013). There was a positive correlation between NPI-caregiver distress with CDR (r=0.363, P=0.045) and ADAS-cog (r=0.449, P=0.019). Conclusion:Based on the observed correlation between IL-1α, as a clinical moiety, and ADAS-cog, as a clinical manifestation of AD, anti-IL-1α therapy in AD could be suggested.

Plasma Cytokines Profile in Subjects with Alzheimer’s Disease: Interleukin 1 Alpha as a Candidate for Target Therapy

Background: Alzheimer’s disease (AD) is the main cause of the neurodegenerative disorder, which is not detected unless the cognitive deficits are manifested. An early prediagnostic specific biomarker preferably detectable in plasma and hence non-invasive is highly sought-after. Various hypotheses refer to AD, with amyloid-beta (Aβ) being the most studied hypothesis and inflammation being the most recent theory wherein pro-and anti-inflammatory cytokines are the main culprits. Materials and Methods: In this study, the cognitive performance of AD patients (n=39) was assessed using mini-mental state examination (MMSE), AD assessment scale-cognitive subscale (ADAS-cog), and clinical dementia rating (CDR). Their neuropsychiatric symptoms were evaluated through neuropsychiatric inventory–questionnaire (NPI-Q). Moreover, plasma levels of routine biochemical markers, pro-/anti-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1 α (IL-1α), IL-1β, IL-2, IL-4, IL-6, IL-8, IL-12p70, IL-10, Interferon-gamma, chemokines, including prostaglandin E2 (PGE-2), monocyte chemoattractant protein-1, interferon gamma-induced protein 10, Aβ peptide species (42, 40) and Transthyretin (TTR) were measured. Results: Our results revealed that Aβ 42/40 ratio and TTR were correlated (r=0.367, P=0.037). IL-1α was directly correlated with ADAS-cog (r=0.386, P=0.017) and Aβ 40 (r=0.379, P=0.019), but was inversely correlated with IL-4 (r=-0.406, P=0.011). Negative correlations were found between MMSE and PGE2 (r=-0.405, P=0.012) and TNF-α/ IL-10 ratio (r=-0.35, P=0.037). CDR was positively correlated with both PGE2 (r=0.358, P=0.027) and TNF-α (r=0.416, P=0.013). There was a positive correlation between NPI-caregiver distress with CDR (r=0.363, P=0.045) and ADAS-cog (r=0.449, P=0.019). Conclusion: Based on the observed correlation between IL-1α, as a clinical moiety, and ADAS-cog, as a clinical manifestation of AD, anti-IL-1α therapy in AD could be suggested. [GMJ.2021;10:e1974]

Copy number variations in primary tumor, serum and lymph node metastasis of bladder cancer patients treated with radical cystectomy

The aim of the present study was to analyze copy number variations (CNV) of multiple oncogenes and tumor suppressor genes in genomic DNA from primary tumor tissue, lymph node metastasis and cell-free DNA (cfDNA) from serum of 72 urothelial carcinoma of bladder (UCB) patients treated with radical cystectomy (RC), using multiplex ligation-dependent probe amplification (MLPA). We hypothesized that primary tumor and lymph node metastasis show similar CNV profiles, and CNV are more present in lymph node metastasis compared to primary tumor tissue. Samples from 43 (59.7%) patients could be analyzed. In total, 35 (83%), 26 (68%) and 8 (42%) patients had CNV in primary tumor, serum and lymph node metastasis, respectively. MYC, CCND1, ERBB2 and CCNE1 displayed the most frequent amplifications. In particular, CNV in ERBB2 was associated with aggressive tumor characteristics. CNV in both ERBB2 and TOP2A were risk factors for disease recurrence. The current findings show that CNV are present in various oncogenes and tumor suppressor genes in genomic DNA from primary tumor, lymph node metastasis and cfDNA from serum. CNV were more present in genomic DNA from primary tumor tissue compared to cfDNA from serum and genomic DNA from lymph node metastasis. Patients with CNV in ERBB2 and TOP2A are at increased risk for disease recurrence following RC. Further studies are necessary to validate, whether these genes may represent promising candidates for targeted-therapy.

Aberrant integrin αv and α5 expression in prostate adenocarcinomas and bone-metastases is consistent with a bone-colonizing phenotype.

BackgroundCollaborative signaling between fibronectin-binding αv and α5 integrins has been implicated in the lethal dissemination of prostate cancer in the bone-metastatic niche, the major source of morbidity and mortality in the disease.MethodsWe assessed the frequency and pattern of expression of these integrins in primary high-grade adenocarcinomas and bone metastases compared to the physiological gland. Formalin-fixed paraffin-embedded (FFPE) radical prostatectomy (RP) samples (n=25) containing ≥ Gleason grade 4 cancer and decalcified surgical or diagnostic bone metastatic samples from 10 patients were stained for integrin αv (ITGAV) and integrin α5 (ITGA5) expression. Antibody optimization and antigen-retrieval was performed beforehand.ResultsITGAV was exclusively expressed in the basal layer of physiological prostate glands whereas αv expression was invariably recapitulated in the malignant gland and bone metastases (100%) in multiple distinct patterns: epithelial membranous, basilar/luminal membranous, punctate cytoplasmic, intense foci as single cells or clusters, and rim stromal layers. The luminal/basilar layer of ITGAV expression was striking in cribriform carcinomas, suggestive of a role in molecular pathogenesis. ITGA5 infrequently highlighted the basal layer of the physiological gland, was absent in primary adenocarcinoma, but was expressed with ITGAV exclusively in bone metastases (71%).ConclusionsWe conclude that ITGAV expression is aberrantly expressed in high frequency in high-grade prostatic adenocarcinomas in patterns suggestive of recapitulated basal cell functions, consistent with a stem-regulatory role that has been proposed. Co-expression and enrichment of αv and α5 in osseous metastases supports their proposed collaborative role in colonization of the bone microenvironment and as candidate targets for therapy.

PB1698 LOSS OF HETEROZYGOSITY IN THE SHORT TANDEM REPEAT LOCI IN THE TUMOR DNA OF THE PATIENTS WITH AML AND ALL: COMPARISON WITH CHROMOSOMAL MICROARRAY DATA

Background:Tumor cell genome aberrations are often detected by various methods. Even while monitoring chimerism after bone marrow transplantation being done for patients with hematological malignancies, one could came across the loss of heterozygosity (LOH) in the loci of short tandem repeats (STR).Aims:To evaluate the frequency of LOH in relapsed and non‐relapsed patients with AML and ALL and possible association of LOH in STR loci with other genomic alterations in malignant cells. terozygosity (LOH) in the loci of short tandem repeats (STR).Methods:This study represents the analysis of STR profiles of 46 patients (27 AML, 17 ALL (4B‐ALL, 8 T‐ALL, 5 Ph + ALL), 2 mixed phenotype acute leukemia) who received allogeneic BMT between 2012 and 2018 in the National Research Center for Hematology, Moscow. The patients were divided in two cohorts: 26 patients without relapses (18AML, 4 Ph + ALL, 3 B‐ALL, 2 T‐ALL, 1 mixed phenotype acute leukemia) and 20 patients having bone marrow or central nervous system relapses (9 AML, 10 ALL, 1 mixed phenotype acute leukemia). DNA samples were isolated from bone marrow in onset of the disease (except six relapsed patients, for whom “debut” samples were not available), in relapse and from cerebrospinal fluid during CNS relapse. The presence of blast cells in bone marrow samples and cerebrospinal fluid was confirmed morphologically and cytogenetically. The control DNA samples were isolated from bone marrow of the same patients in remission. STR‐profiles were assessed by PCR with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR‐markers and amelogenin loci (Gordiz Ltd, Russia). The frequencies of heterozygosity for STR loci D3S1358, TH01, D12S391, D1S1656, D10S1248, D22S1045, D2S441, D7S820, D13S317, FGA, TPOX, D18S51, D16S539, D8S1179, CSF1PO, D5S818, VWA, D21S11, SE33 was evaluated on extended patients and donors groups, 120 persons each. The fragment analysis was performed on ABI3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4‐0 software. Whole‐genome chromosomal microarray (CMA) was performed on Affimetrix CytoscanTM HD array platform. Data processing was carried out using Chromosome Analysis Suite v.4.0 software, genome assembly version hg19, GRCh37 Genome Reference Consortium Human Reference 37, https://genome.ucsc.edu/.Results:In preliminary experiments on extended cohorts the frequency of heterozygosity for each of STR loci was established between 70% to 95%. Three patients (11.5%) were identified with a loss of heterozygosity at the STR loci in the “no relapses” cohort, 2 from 18 AML (11%) and one T‐ALL patient. For “patients in relapse” LOH by STR analysis was detected in 10 cases from 20, and additionally three patients were identified as LOH‐positive by CMA (65%): 5 from 9 AML patients (55.5%) and 7 from10 ALL patients (70%) and one mixed phenotype acute leukemia patient (Table1). One B‐ALL patient with four CNS relapses and three bone marrow relapses had LOH in 10 from 15 heterozygous loci (Table1, patient#3). The CMA showed that LOH of STR loci often is a result of uniparental disomy (UPD). For two AML relapsed patients (Table1, patients#2,#5) UPD of 13 chromosome resulted into FLT3/ITD mutation (13q12.11) homozygocity. Therefore these patients shoud be considered candidates for target therapy against FLT3.Summary/Conclusion:LOH frequency in STR loci is significantly higher for relapsed patients versus those relapse‐free. CMA confirmes and expands STR analysis data,evaluates genes crucial for disease progression in certain patient and may possibly help to select effective target therapy.image

SPOCK1 contributes to the third-generation EGFR tyrosine kinase inhibitors resistance in lung cancer.

Explanation of the mechanism of resistance to third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and the development of a novel strategy for drug resistance are imperative in third-generation EGFR-TKIs-resistant non-small cell lung cancer (NSCLC). SPOCK1 was found to be abnormally expressed in various tumors including lung cancer, however, there was no study focused on the role of SPOCK1 in third-generation EGFR-TKIs resistant lung cancer cells. We investigated the roles of SPOCK1 in NSCLC with third-generation EGFR-TKIs resistance. We showed that SPOCK1 was upregulated in the osimertinib-resistant lung cancer cells and knockdown of SPOCK1 inhibits osimertinib-resistant cells growth and overcomes resistance. Furthermore, we demonstrated that the SPOCK1 was higher in clinical NSCLC specimens compared with the normal lung tissues, and the higher expression of SPOCK1 correlated with poor prognosis. In addition, the overexpression of SPOCK1 in NSCLC tissues was positively correlated with MMP11 and TGFβ1. Our study suggested that SPOCK1 could be an independent prognostic factor in NSCLC and would be a candidate for target therapy in osimertinib-resistant lung tumors.

Cannabinoid Receptors and CXCR4 Cross-Talk in Lymphoma Cells

Background: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with a high rate of relapses after therapy. Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with varied outcome. For both diseases there is a need for new therapies. Cannabinoid receptors (CBs), which are overexpressed in most cases of MCL and CLL compared to normal B cells (Islam et al., 2003; Gustafsson et al., 2008; Freund et al., 2016) are promising novel therapeutic targets. CBs are membrane-bound receptors that convey signals from the microenvironment to the cells. There are two types of CBs: CB1 and CB2. CB1 is suggested to be involved in retention and/or egress of MCL cells from the tissue to the blood circulation (Wasik et al., 2014). CB2 is expressed by normal B-cells where it regulates positioning and retention of cells in tissue (Pereira et al., 2009; Basu et al., 2011; Muppidi et al., 2011) and in pre-B-cell acute lymphoblastic leukemia, involved in the energy metabolism (Chan et al., 2017). The retention/egress of the B-cell lymphoma cells is mainly regulated by chemokine receptors and adhesion molecules. The chemokine receptor CXCR4 is one of the most highly expressed chemokine receptors in MCL and CLL. 2-arachidonoylglycerol (2-AG, CB1/CB2 endogenous ligand) and CXCL12 (CXCR4 ligand) are synthetized and secreted by stromal cells in the bone marrow (Kose et al., 2018; Burger and Gribben, 2014). The endocannabinoids levels in cancer are suggested to have a role in cancer progression (Sailler et al., 2014) while CXCL12 is already a candidate target for therapy using a CXCR4 inhibitor AMD3100.Aim: To investigate a possible crosstalk between CBs and CXCR4 in MCL and CLL cells.Methods: Patients with newly diagnosed MCL (n=8) or CLL (n=25) gave informed consent to participate in the study. Lymphoma cells were enriched by negative selection. Fifteen primary lymphoma samples and the JeKo MCL cell line were subjected to chemotaxis towards CXCL12 and/or 2-AG. CXCR4 membrane expression was assessed by flow cytometry. Selective CB1 and CB2 antagonists were used to investigate the underlying mechanisms. CB1, CB2 and CXCR4 encoding genes levels were measured by qPCR and normalized to B cells from tonsil.Results and Conclusion: 2-AG induced chemotaxis in 11/15 MCL and CLL samples. In JeKo, 2-AG-induced migration was blocked by a CB2 antagonist, suggesting that signaling via CB2 is involved. When the primary cells were subjected to migration towards CXCL12, two patterns of chemotaxis were observed. The first pattern was seen in 7/15 samples that migrated towards CXCL12. In these samples, the migration was inhibited when 2-AG was combined with CXCL12. The second type of response was observed in 8/15 samples, those samples did not migrate towards CXCL12 but chemotaxis was enhanced by combining 2-AG and CXCL12. MCL and CLL samples expressed variable mRNA levels of CB1 (RFI range: 0.0-204) and CB2 (RFI range: 0.8-14.3) and all expressed CXCR4 at mRNA (RFI range: 0.1-215.8) and protein (MFI range: 1278-19301) levels that did not differ neither between the two diseases nor between the two migratory groups. When all 15 samples were combined, CB1 mRNA levels, but not CB2 mRNA, correlated to the chemotaxis towards CXCL12 (Spearman correlation coefficient = 0.626; p=0.01). In contrast, CB2 mRNA levels, but not CB1, correlated to chemotaxis towards 2-AG (Spearman correlation coefficient = 0.532; p=0.04), which is in agreement with the effects observed in JeKo. Furthermore, CB1 and CB2 mRNA levels correlated to chemotaxis towards the combination of CXCL12 and 2-AG both (for CB1 mRNA: Spearman correlation coefficient= 0.588; p=0.02 and for CB2 mRNA: 0.589; p=0.02). Neither CXCL12-induced CXCR4 receptor internalization, nor recycling was influenced by 2-AG incubation. Our findings indicate a novel pathway regulating chemotaxis of MCL and CLL implicating a cross-talk between CBs and CXCR4. The fact that the capacity to internalize CXCR4 remained intact after incubation with 2-AG suggests that the reduced CXCL12-mediated migration when 2-AG was combined could be due to an impaired downstream signaling in lymphoma cells.Importance: Lymphoma cells residing in the tissue receive pro-survival stimuli and are protected from chemotherapy by signals from the microenvironment. A better understanding of how lymphoma cell migration and tissue retention are regulated can be a step towards more efficient therapies. DisclosuresWahlin:Gilead: Consultancy, Honoraria, Research Funding; Roche: Research Funding.

Abstract A026: Expression of fibroblast growth factor receptor 1 isoforms and activation of different pathways in prostate cancer progression

Abstract Bone metastases typically develop in patients with advanced prostate cancer (PCa). Androgen deprivation is commonly used as treatment, but responses are short, and eventually the disease progresses to a castration-resistant form (CRPC). The fibroblast growth factor (FGF) axis is commonly altered during PCa progression. Our group and others have implicated the FGF axis in the pathogenesis of PCa progression in bone, identified it as a candidate target for therapy, and suggested that FGF receptor (FGFR)-1 mediates a positive feedback loop between PCa cells and bone cells. RNA sequencing studies of 183 human PCa samples indicated that the mean expression of FGFR-1 is the highest of all the FGFR family genes studied. Analyses of these FGFR-1 transcripts identified eight different protein-coding transcripts to be the most abundantly expressed with diverse human PCa tissue samples expressing different FGFR-1 isoforms. The overall goal of this project is to investigate implications of FGFR1/FGFR1 isoforms expression in the pathogenesis of PCa bone metastases. When we assessed the expression of the two best-characterized FGFR-1 isoforms (alpha (NM_023110.2) and beta (NM_023105.2), which represent the most abundant protein coding transcripts found in PCa, we observed that all patient-derived xenografts (PDXs) studied express significantly higher levels of FGFR-1 alpha than beta while PCa cell lines mostly express the beta isoform. Mining The Cancer Genome Atlas (TCGA) PCa data, we identified distinct patterns of gene expression associated with each FGFR-1 isoform (alpha and beta). Briefly, FGFR-1 beta (but not alpha) is significantly associated with pathways including the MAP-kinase signaling cascade, signaling by FGFR in disease and pathways in cancer. In vitro studies of FGF signaling activation in PCa cells expressing FGFR-1 isoforms alpha, beta or empty vector, confirmed these results. Male SCID mice injected intracardially with PC3-FGFR-1 alpha exhibited higher death rates compared to animals injected with either PC3-FGFR-1 beta or PC3-empty vector. Furthermore, higher number of metastases per mice as well as higher number of mice with bone metastases were observed in animals injected with PC3-FGFR-1 alpha. The fact that different prostate tumors express different FGFR-1 isoforms suggests that FGFR1 alpha and beta isoforms activate different genes or pathways in PCa cells and this may underlie, at least in part, PCa heterogeneity, pattern of progression, and differences in response to FGFR1 inhibitors. Further studies will warrant the understanding of the implications of FGFR-1 isoforms expression in the pathogenesis of PCa. Citation Format: Estefania Labanca, Jun Yang, Peter Shepherd, Justin Roberts, Michael Starbuck, Bradley Broom, Matthew Iyer, Christopher Logothetis, Arul Chinnaiyan, Nora Navone. Expression of fibroblast growth factor receptor 1 isoforms and activation of different pathways in prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A026.

Mutational landscape and clonal diversity of pulmonary adenoid cystic carcinoma

ABSTRACTPulmonary adenoid cystic carcinoma is a rare and indolent lung malignancy, characterized by a protracted but unpredictable growth behavior. Currently, the treatment of PACC relies on surgery and local radiotherapy. However, treatment options for advanced PACC patients are limited. A larger number of studies demonstrated that advanced PACC patients obtained limited benefit from chemotherapy. Moreover, only a few case reports revealed PACC patients were candidates for target therapy. Therefore, there is an urgent need to develop novel therapies. Due to its rareness, its mutational landscape remains largely elusive. In this study, we performed capture-based ultra-deep sequencing on multiregional surgical specimens obtained from 8 PACC patients using a panel consisting of 295 cancer-related genes. Our data revealed distinctive mutational spectrum of PACC, which differed from non-small cell lung cancer and adenoid cystic carcinomas originated from other anatomical sites. PACC, lacking mutations in a majority of non-small cell lung cancer driver genes, has frequent mutations in genes participating in chromatin remodeling and NOTCH signaling pathway. We also elucidated spatial intra-tumoral heterogeneity, which varied among cases. Most mutations in chromatin remodelers were subclonal. Collectively, our findings elucidated molecular signature associated with PACC and highlighted the potential for epigenetic therapy in this disease.

Abstract A28: Targeted therapy of Ewing sarcoma

Abstract Ewing sarcoma is a high-risk sarcoma with overall 50% survival, unchanged for decades. No effective new therapies have been introduced in that time frame. It is thus an ideal candidate for targeted therapy. It is also a tumor with several candidate targets for therapy. Since essentially all Ewing sarcomas express the cell surface antigen CD99, monoclonal antibodies can be used to deliver nanoparticle-encapsulated chemotherapeutic agents with high efficiency. Compared to conventional antibody-drug conjugates (ADCs), binding avidity and delivered dose per bound antibody is increased exponentially. Similarly, the tumor-specific EWS-FLI1 or equivalent gene translocation that drives oncogenesis is an attractive candidate target, so far untargetable with conventional small molecules. Both nanoparticle-delivered shRNA and CRISPR/Cas9 constructs targeting the fusion gene are candidate therapeutics that can be directed specifically to the fusion junction or the ets domain. Downstream targets of the fusion gene include a number of coding and noncoding genes, among them FEZF1-AS, a long noncoding RNA (lncRNA) that we have shown is both a direct target of the Ewing fusion gene and an oncogenic driver of tumor growth, invasion, and metastasis. While otherwise untargetable, lncRNAs can be effectively downregulated by ASOs (anti-sense oligonucleotides) with a proportional attenuation of their oncogenic effect. Here we present evidence that a novel CD99 targetable nanoparticle, termed hybrid polymerized liposomal nanoparticles, can effectively deliver small-molecule cytotoxics with complete tumor ablation in a nude mouse model of Ewing sarcoma, as well as EWS-FLI1 targeted CRISPR/Cas9 constructs and FEZF1-AS targeted ASOs, with proportional knockdown and oncogenic attenuation. Tumor uptake of the therapeutic nanoparticles is rapid, with over 90% of particles at therapeutic doses bound and internalized within 20 minutes in vitro. The IC50 for cytotoxics is improved 40-fold over free agent and 20-fold over untargeted nanoparticle formulations. Animals with Ewing sarcoma xenografts treated with CD99-targeted hPLNs containing irinotecan show complete tumor ablation with no tumor regrowth at 90 days, 20 days after cessation of treatment, unlike free irinotecan or untargeted irinotecan liposomal nanoparticle animals, all of whom died with progressive tumor growth. Surviving animals showed no toxicity. Ancillary studies identified no evidence of bone marrow, liver, or kidney toxicity at therapeutic doses. Over 70% of tumor cells express EGFP after internalizing targeted nanoparticles containing CRISPR/Cas9 plasmids with an EGFP construct. Similarly, FEZF1-AS lncRNA expression is reduced by over 70% after treatment with FEZF1-AS specific ASOs. Taken together, these results suggest that both targetable (cytotoxics delivered preferentially to tumor cells) and previously untargetable features (fusion genes, lncRNAs) can be effectively and specifically treated when conventional and novel therapeutics are delivered directly to tumor cells by CD99-targeted nanoparticles. The successful treatment of Ewing sarcoma xenografts and metastases in animal models by any of these approaches also suggests combined therapy with both conventional and unconventional agents is feasible, with no appreciable toxicity. Citation Format: Timothy J. Triche, Jon Nagy, Hyung Kang, Chris Denny, Sheetal Bajaj Mitra. Targeted therapy of Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A28.

The implication from RAS/RAF/ERK signaling pathway increased activation in epirubicin treated triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is not sensitive to RAS/RAF/ERK signaling pathway (ERK pathway) targeting therapy, due to the absence of excessive activation of ERK pathway. However, the kinase cascades might be activated after chemotherapy in TNBC. Here we aimed to predict whether ERK pathway targeting therapy could be used as an adjuvant therapy in TNBC.MethodsWithin online GEO datasets (GSE43816 and GSE54326), gene set enrichment analysis (GSEA) was performed to detect molecular changes in epirubicin treated TNBC samples and cells, ERK pathway components and regulation genes changes were included.ResultsIn epirubicin treated TNBC samples and cells, we found ERK pathway components (eg. MAPK13, MAP3K1, MAPK12, MAPK11 and MAPKAPK3) were obviously enriched, also, expression of ERK pathway positive regulation genes significantly increased (P<0.05) and negative regulation genes decreased (P<0.05) in epirubicin resistant cells. Moreover, phosphorylated ERK levels were significantly elevated in MDA-MB-231 cells after epirubicin treatment.ConclusionERK signaling pathway was more activated in epirubicin treated TNBC, possibly contributing to the epirubicin resistance in TNBC, it implicated that ERK pathway could be used as an novel candidate for targeting therapy in refractory and relapse TNBC.

Oncogenic role and therapeutic targeting of ABL-class and JAK-STAT activating kinase alterations in Ph-like ALL.

New therapies for Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) patients are urgently needed. The genetic landscape of Ph-like ALL is characterized by a diverse array of kinase-activating alterations (including rearrangements, sequence mutations, and copy number alterations), suggesting that patients with Ph-like ALL are candidates for targeted therapy, similar to BCR-ABL1 ALL. We sought to investigate the functional role and targetability of the spectrum of kinase-activating alterations identified in Ph-like ALL. We demonstrate cytokine-independent growth and activation of JAK-STAT signaling pathways in Ba/F3 cells by all alterations tested. The development of murine Arf-/- pre-B ALL expressing RCSD1-ABL2 or SSBP2-CSF1R was accelerated with the presence of IK6, a dominant negative isoform of Ikaros common in Ph-like ALL, providing evidence that these fusions are leukemogenic. In vitro screening using a panel of tyrosine kinase inhibitors against 14 different kinase alterations identified the ABL1-inhibitor, dasatinib, as a potent inhibitor of ABL-class fusions (ABL1, ABL2, CSF1R, PDGFRB), whereas the JAK1/JAK2 inhibitor ruxolitinib, was most effective against JAK-STAT-activating alterations (JAK1, JAK2, JAK3, IL7R, IL2RB), but not TYK2. Evaluation of dasatinib or ruxolitinib against patient-derived xenograft models demonstrated superior antileukemic efficacy when combined with dexamethasone compared with either agent alone. These data provide the foundation for rationally designed clinical trials that assess the efficacy of targeted therapy in patients with Ph-like ALL.

PHF21B overexpression promotes cancer stem cell-like traits in prostate cancer cells by activating the Wnt/\u03b2-catenin signaling pathway

BackgroundPHF21B is newly identified to be involved in the tumor progression; however, its biological role and molecular mechanism in prostate cancer have not been defined. This study is aimed to study the role of PHF21B in the progression of prostate cancer.MethodsReal-time PCR, immunohistochemistry and western blotting analysis were used to determine PHF21B expression in prostate cancer cell lines and clinical specimens. The role of PHF21B in maintaining prostate cancer stem cell-like phenotype was examined by tumor-sphere formation assay and expression levels of stem cell markers. Luciferase reporter assay, western blot analysis, enzyme-linked immunosorbent assay and ChIP assay were used to determine whether PHF21B activates the Wnt/β-catenin signaling by transcriptionally downregulating SFRP1 and SFRP2.ResultsOur results revealed that PHF21B was markedly upregulated in prostate cancer cell lines and tissues. High PHF21B levels predicted poorer recurrence-free survival in prostate cancer patients. Gain-of-function and loss-of-function studies showed that overexpression of PHF21B enhanced, while downregulation suppressed, the cancer stem cell-like phenotype in prostate cancer cells. Xenograft tumor model showed that silencing PHF21B decreased the ability of tumorigenicity in vivo. Notably, Wnt/β-catenin signaling was hyperactivated in prostate cancer cells overexpressing PHF21B, and mediated PHF21B-induced cancer stem cell-like phenotype. Furthermore, PHF21B suppressed repressors of the Wnt/β-catenin signaling cascade, including SFRP1 and SFRP2. These results demonstrated that PHF21B constitutively activated wnt/β-catenin signaling by transcriptionally downregulating SFRP1 and SFRP2, which promotes prostate cancer stem cell-like phenotype.ConclusionsOur results revealed that PHF21B functions as an oncogene in prostate cancer, and may represent a promising prognostic biomarker and an attractive candidate for target therapy of prostate cancer.

Immunohistochemical c-kit expression in uterine serous carcinoma tissue.

Uterine serous carcinoma (USC) is an aggressive tumor that represents only 10% of endometrial cancer cases but accounts for a disproportionate number of deaths due to uterine cancer. Advances in the development of specific c-kit receptor-targeted drugs have promoted its potential therapeutic application as a target in tumor-related diseases. The aim of the present study was to evaluate imunohistochemical expression of c-kit in USC tissue in order to assess whether positive cases can be candidates for targeted therapy. C-kit expression assessment by immunohistochemistry was performed on deparaffinized sections of paraffin-embedded tissue blocks of confirmed consecutive available USC uterine specimens of patients diagnosed from 2000 to 2014. Sections of gastrointestinal stromal tumor (GIST) tissue known to contain c-kit served as positive controls. Immunohisto- chemical c-kit staining was not observed in any of 31 USC tissue samples examined. Intense staining was observed in the sections of GIST tissue. The present results may indicate that primary USC is not a candidate for c-kit targeted therapy.

Alox5 Blockade Eradicates JAK2V617F-Induced Polycythemia Vera in Mice.

Myeloproliferative neoplasms such as polycythemia vera (PV), which are associated with the JAK mutation V617F, remain incurable despite progress in the use of JAK2 inhibitors for treatment of some of these diseases. In this study, we employed mice that undergo JAK2V617F-induced PV as a tool to explore new candidate targets for therapy. Our investigations focused on the lipid metabolic enzyme arachidonate 5-lipoxygenase (Alox5), which we found to be strongly upregulated by JAK2V617F in hematopoietic cells in vitro and in vivo Notably, genetic deletion of Alox5 or its inhibition in mice with a bioactive small-molecule inhibitor was sufficient to attenuate PV development. This therapeutic effect was associated with induction of a blockade in cell-cycle progression and also with apoptosis in PV cells. Genetic loss exerted an inhibitory effect on PV-initiating cells. Similarly, Alox5 inhibition was sufficient to suppress colony formation in human JAK2V617F-expressing CD34+ cells. Mechanistic investigations showed that Alox5 inhibition reduced AKT activation and decreased β-catenin expression in JAK2V617F-expressing cells. Together, our results define Alox5 as a key genetic effector of JAK2V617F in driving PV, and they identify this enzyme as a candidate therapeutic target to treat this refractory myeloproliferative neoplasm. Cancer Res; 77(1); 164-74. ©2016 AACR.

Oncofetal Chondroitin Sulfate Glycosaminoglycans Are Key Players in Integrin Signaling and Tumor Cell Motility.

The cancer-specific expression of ofCS aids in metastatic phenotypes and is a candidate target for therapy. Mol Cancer Res; 14(12); 1288-99. ©2016 AACR.

NOX-driven ROS formation in cell transformation of FLT3-ITD-positive AML.

In different types of myeloid leukemia, increased formation of reactive oxygen species (ROS) has been noted and associated with aspects of cell transformation, including the promotion of leukemic cell proliferation and migration, as well as DNA damage and accumulation of mutations. Work reviewed in this article has revealed the involvement of NADPH oxidase (NOX)-derived ROS downstream of oncogenic protein-tyrosine kinases in both processes, and the related pathways have been partially identified. FMS-like tyrosine kinase 3 with internal tandem duplications (FLT3-ITD), an important oncoprotein in a subset of acute myeloid leukemias, causes activation of AKT and, subsequently, stabilization of p22phox, a regulatory subunit for NOX1-4. This process is linked to ROS formation and DNA damage. Moreover, FLT3-ITD signaling through STAT5 enhances expression of NOX4, ROS formation, and inactivation of the protein-tyrosine phosphatase DEP-1/PTPRJ, a negative regulator of FLT3 signaling, by reversible oxidation of its catalytic cysteine residue. Genetic inactivation of NOX4 restores DEP-1 activity and attenuates cell transformation by FLT3-ITD invitro and invivo. Future work is required to further explore these mechanisms and their causal involvement in leukemic cell transformation, which may result in the identification of novel candidate targets for therapy.