Tumor-infiltrating lymphocytes are related to positive clinical prognoses in numerous cancer types. Programmed death ligand 1 (PD-L1), a mediator of the PD-1 receptor, plays an inhibitory role in cancer immune responses. PD-L1 upregulation can impede infiltrating T-cell functions in lung adenocarcinoma (LUAD), a lung cancer subtype. However, associations between the expression of PD-L1 and infiltration of B cells (a major immunoregulatory cell) remain unknown. Therefore, we investigated the role of infiltrating B cells in LUAD progression and its correlation with PD-L1 expression. The Cancer Genome Atlas (TCGA) LUAD data set was used to explore associations among B-cell infiltration, PD-L1 expression, clinical outcome, and gene landscape. Gene set enrichment analysis was used to explore putative signaling pathways and candidate genes. The drug enrichment analysis was used to identify candidate genes and the related drugs. We found that high B-cell infiltration was correlated with better prognoses; however, PD-L1 may interfere with the survival advantage in patients with high B-cell infiltration. The gene landscape was characterized comprehensively, with distinct PD-L1 levels in cell populations with high B-cell infiltration. We obtained five upregulated signaling pathways from the gene landscape: apoptosis, tumor necrosis factor (TNF)-α signaling via nuclear factor (NF)-κB, apical surface, interferon-α response, and KRAS signaling. Moreover, four candidate genes and their related target drugs were also identified, namely interleukin-2β receptor (IL2RB), IL-2γ receptor (IL2RG), Toll-like receptor 8 (TLR8), and TNF. These findings suggest that tumor-infiltrating B cells could act as a clinical factor in anti-PD-L1 immunotherapy for LUAD.

Lung cancer remains one of the leading causes of cancer-related deaths. Non-small cell lung cancer (NSCLC) is the most common histologic type of lung cancer. Medical and scientific progress has led to longer survival in an increasing number of patients suffering from cancer. Concerning patients with advanced NSCLC, there is a subgroup with long-term survival. The human epidermal growth factor receptor (EGFR) family plays a key role in tumor development. This cluster of genes is associated with augmented angiogenesis and enhanced proliferation, survival, and migration of tumor cells. The CIMAvax-EGF vaccine consists of a chemical conjugate of the EGF with the P64 protein derived from the Meningitis B bacteria and the Montanide ISA 51, as adjuvant. The vaccine induces antibodies against EGF that results in EGF withdrawal. CIMAvax-EGF has been demonstrated to be safe and immunogenic in advanced NSCLC patients. Here we summarize the current knowledge of the mechanism of action of CIMAvax-EGF, highlighting the impact of this anti-EGF-based vaccine on the long-term survival of advanced NSCLC patients.

Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-Prkdc

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal antigen expressed on multiple tumors and has no significant expression on normal human tissues. ROR1 is highly upregulated in chronic lymphocytic leukemia (CLL) B cells. NOD-scid IL2rg

BACKGROUND: This study aimed to screen sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer.
METHODS: In this study, Illumina digital gene expression sequencing technology was applied and differentially expressed genes (DEGs) between patients presenting pathological complete response (pCR) and non-pathological complete response (NpCR) were identified. Further, gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were then performed. The genes in significant enriched pathways were finally quantified by quantitative real-time PCR (qRT-PCR) to confirm that they were differentially expressed. Additionally, GSE23988 from Gene Expression Omnibus database was used as the validation dataset to confirm the DEGs.
RESULTS: After removing the low-quality reads, 715 DEGs were finally detected. After mapping to KEGG pathways, 10 DEGs belonging to the ubiquitin proteasome pathway (HECTD3, PSMB10, UBD, UBE2C, and UBE2S) and cytokine-cytokine receptor interactions (CCL2, CCR1, CXCL10, CXCL11, and IL2RG) were selected for further analysis. These 10 genes were finally quantified by qRT-PCR to confirm that they were differentially expressed (the log
CONCLUSION: Our results suggested that these 10 genes belonging to these two pathways might be useful as sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer.

Severe combined immunodeficiency is an inherited disease with profoundly defective T cells, B cells, and natural killer cells. X-linked severe combined immunodeficiency is the most common form. In this report, we describe a 4-month-old male infant who was admitted to our hospital with progressive breathlessness and abdominal mass. He was diagnosed with hepatoblastoma and presented a pneumocystis jirovecii pneumonia at the beginning of chemotherapy. Definitive diagnosis of X-linked severe combined immunodeficiency was established by DNA analysis of the interleukin 2 receptor gamma chain gene. This case is the first report which describes an X-linked severe combined immunodeficiency patient with hepatoblastoma.

OBJECTIVE: As the current therapeutic strategies for human hepatocellular carcinoma (HCC) have been proven to have limited effectiveness, immunotherapy becomes a compelling way to tackle the disease. We aim to provide humanised mouse (humice) models for the understanding of the interaction between human cancer and immune system, particularly for human-specific drug testing.
DESIGN: Patient-derived xenograft tumours are established with type I human leucocyte antigen matched human immune system in NOD-
RESULTS: Similar to the clinical outcomes, the human immune system in our model is educated by the tumour and exhibits exhaustion phenotypes such as a significant declination of leucocyte numbers, upregulation of exhaustion markers and decreased the production of human proinflammatory cytokines. Notably, cytotoxic immune cells decreased more rapidly compared with other cell types. Tumour infiltrated T cells have much higher expression of exhaustion markers and lower cytokine production compared with peripheral T cells. In addition, tumour-associated macrophages and myeloid-derived suppressor cells are found to be highly enriched in the tumour microenvironment. Interestingly, the tumour also changes gene expression profiles in response to immune responses by upregulating immune checkpoint ligands. Most importantly, in contrast to the NSG model, our model demonstrates both therapeutic and side effects of immune checkpoint inhibitors pembrolizumab and ipilimumab.
CONCLUSIONS: Our work provides a model for immune-oncology study and a useful parallel-to-human platform for anti-HCC drug testing, especially immunotherapy.

The blockade of immune checkpoints by anti-receptor and/or anti-ligand mAb is one of the most promising approaches to cancer immunotherapy. The interaction between Ig-like transcript 3 (ILT3), a marker of tolerogenic dendritic cells, also known as LILRB4/LIR5/CD85k, and its still unidentified ligand on the surface of activated human T cells is potentially important for immune checkpoint blockade. To identify the ILT3 ligand, we generated mAb by immunizing mice with Jurkat acute T cell leukemia, which binds ILT3.Fc to its membrane. Flow cytometry, mass spectrometry, and Biacore studies demonstrated that the ILT3 ligand is a CD166/activated leukocyte cell adhesion molecule. Knockdown of CD166 in primary human T cells by nucleofection abolished the capacity of ILT3.Fc to inhibit CD4

Previous studies have proposed that epithelial to mesenchymal transition (EMT) in breast cancer cells regulates metastasis, stem cell properties and chemo-resistance; most studies were based on in vitro culture of cell lines and mouse transgenic cancer models. However, the identity and function of cells expressing EMT-associated genes in normal murine mammary gland homeostasis and human breast cancer still remains under debate. Using in vivo lineage tracing and triple negative breast cancer (TNBC) patient derived xenografts we demonstrate that the repopulating capacity in normal mammary epithelial cells and tumorigenic capacity in TNBC is independent of expression of EMT-associated genes. In breast cancer, while a subset of cells with epithelial and mesenchymal phenotypes have stem cell activity, in many cells that have lost epithelial characteristics with increased expression of mesenchymal genes, have decreased tumor-initiating capacity and plasticity. These findings have implications for the development of effective therapeutic agents targeting tumor-initiating cells.

Combining natural killer (NK) cell adoptive transfer with hypomethylating agents (HMAs) is an attractive therapeutic approach for patients with acute myeloid leukemia (AML). However, data regarding the impact of HMAs on NK cell functionality are mostly derived from in vitro studies with high nonclinical relevant drug concentrations. In the present study, we report a comparative study of azacitidine (AZA) and decitabine (DAC) in combination with allogeneic NK cells generated from CD34

MicroRNA-138 (miR-138) is a pro-survival oncomiR for glioma stem cells. In malignant gliomas, dysregulated expression of microRNAs, such as miR-138, promotes Tumour initiation and progression. Here, we identify the ancillary role of the CCAAT/enhancer binding protein β (C/EBPβ) as a transcriptional activator of miR-138. We demonstrate that a short 158 bp DNA sequence encoding the precursor of miR-138-2 is essential and sufficient for transcription of miR-138. This short sequence includes the A-box and B-box elements characteristic of RNA Polymerase III (Pol III) promoters, and is also directly bound by C/EBPβ via an embedded 'C/EBPβ responsive element' (CRE). CRE and the Pol III B-box element overlap, suggesting that C/EBPβ and transcription factor 3C (TFIIIC) interact at the miR-138-2 locus. We propose that this interaction is essential for the recruitment of the RNA Pol III initiation complex and associated transcription of the oncomiR, miR-138 in malignant gliomas.

Antibody therapy constitutes a major advance in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To evaluate the efficacy and the mechanisms of action of CD19 monoclonal antibody therapy in pediatric BCP-ALL, we tested an Fc-engineered CD19 antibody carrying the S239D/I332E mutation for improved effector cell recruitment (CD19-DE). Patient-derived xenografts (PDX) of pediatric mixed-lineage leukemia gene (

BACKGROUND & AIMS: Drugs that inhibit the erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2) are the standard treatment of patients with different types of cancer, including HER2-overexpressing gastroesophageal tumors. Unfortunately, cancer cells become resistant to these drugs, so overall these drugs provide little benefit to patients with these tumors. We investigated mechanisms that mediate resistance of esophageal adenocarcinoma (EAC) cells and patient-derived xenograft tumors to ERBB inhibitors.
METHODS: We cultured primary tumor cells, isolated from EAC patient samples, and OE19 and OE33 EAC cell lines with trastuzumab (an inhibitor of HER2), with or without pertuzumab (which inhibits dimerization of HER2 with HER3) or a specific antibody against HER3 (anti-HER3). HER2 was knocked down by expression of small hairpin RNAs. In addition, cells were incubated with NRG1-β, a mediator of HER2-HER3 signaling, or A83-01, an inhibitor of transforming growth factor beta (TGFβ) signaling. Cells were analyzed for markers of the epithelial to mesenchymal transition (EMT) using flow cytometry, immunofluorescence, and quantitative reverse transcription polymerase chain reaction. We performed limiting dilution, transwell, and cell viability assays to study the functional effects of HER2 and HER3 inhibition and reactivation. We analyzed publicly available EAC gene expression datasets to correlate expression of ERBB genes with genes encoding epithelial and mesenchymal proteins. NOD.Cg-Prkdc
RESULTS: EAC cells incubated with trastuzumab decreased expression of epithelial markers (CD24, CD29, and CDH1) and increased expression of mesenchymal markers (CXCR4, VIM, ZEB1, SNAI2, and CDH2), compared with cells not exposed to trastuzumab, indicating induction of EMT. Addition of NRG1-β to these cells restored their epithelial phenotype. Incubation of EAC cells with trastuzumab and pertuzumab accelerated the expression of EMT markers, compared with cells incubated with trastuzumab alone. EAC cells cultured for 2 months with a combination of trastuzumab and pertuzumab became resistant to chemotherapeutic agents (5-fluoruracil, carboplatin, cisplatin, eribulin, and paclitaxel), based on their continued viability, which was accompanied with an enhanced migratory capacity in transwell assays and clonogenicity in limiting dilution analyses. In comparisons of EAC gene expression patterns, we associated high expression of ERBB3 with an epithelial gene expression signature; expression of TGFβ correlated with expression of EMT-related genes, and we found an inverse correlation between expression of TGFB1 and ERBB3. EAC cells incubated with ERBB inhibitors began to secrete ligands for the TGFβ receptor and underwent EMT. Incubation of EAC cells with trastuzumab, followed by 10 days of incubation with the TGFβ receptor inhibitor in the presence of trastuzumab, caused cells to regain an epithelial phenotype. EAC patient-derived xenograft tumors grew more slowly in mice given the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor than in mice given single agents or a combination of trastuzumab and pertuzumab. Tumors exposed to trastuzumab and pertuzumab expressed EMT markers and were poorly differentiated, whereas tumors exposed to the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor expressed epithelial markers and were more differentiated.
CONCLUSIONS: EAC cells become resistant to trastuzumab and pertuzumab by activating TGFβ signaling, which induces EMT. Agents that block TGFβ signaling can increase the anti-tumor efficacies of trastuzumab and pertuzumab.

The therapeutic potential of CRISPR system has already been demonstrated in many instances and begun to overlap with the rapidly expanding field of cancer immunotherapy, especially on the production of genetically modified T cell receptor or chimeric antigen receptor (CAR) T cells. Efficient genomic disruption of multiple gene loci to generate universal donor cells, as well as potent effector T cells resistant to multiple inhibitory pathways such as PD-1 and CTLA4 is an attractive strategy for cell therapy. In this study, we accomplished rapid and efficient multiplex genomic editing, and re-directing T cells with antigen specific CAR via a one-shot CRISPR protocol by incorporation of multiple gRNAs in a CAR lentiviral vector. High efficient double knockout of endogenous TCR and HLA class I could be easily achieved to generate allogeneic universal CAR T cells. We also generated Fas-resistant universal CAR T cells by triple gene disruption. Simultaneous gene editing of four gene loci using the one-shot CRISPR protocol to generate allogeneic universal T cells deficient of both PD1 and CTLA-4 was also attempted.

There is currently a paucity of preclinical models available to study the metastatic process in esophageal cancer. Here we report FLO-1, and its isogenic derivative FLO-1LM, as two spontaneously metastatic cell line models of human esophageal adenocarcinoma. We show that FLO-1 has undergone epithelial-mesenchymal transition and metastasizes following subcutaneous injection in mice. FLO-1LM, derived from a FLO-1 liver metastasis, has markedly enhanced proliferative, clonogenic, anti-apoptotic, invasive, immune-tolerant and metastatic potential. Genome-wide RNAseq profiling revealed a significant enrichment of metastasis-related pathways in FLO-1LM cells. Moreover, CDH1, which encodes the adhesion molecule E-cadherin, was the most significantly downregulated gene in FLO-1LM compared to FLO-1. Consistent with this, repression of E-cadherin expression in FLO-1 cells resulted in increased metastatic activity. Importantly, reduced E-cadherin expression is commonly reported in esophageal adenocarcinoma and independently predicts poor patient survival. Collectively, these findings highlight the biological importance of E-cadherin activity in the pathogenesis of metastatic esophageal adenocarcinoma and validate the utility of FLO-1 parental and FLO-1LM cells as preclinical models of metastasis in this disease.

Systemic mastocytosis are rare neoplasms characterized by accumulation of mast cells in at least one internal organ. The majority of systemic mastocytosis patients carry KIT D816V mutation, which activates constitutively the KIT receptor. Patient with advanced forms of systemic mastocytosis, such as aggressive systemic mastocytosis or mast cell leukemia, are poorly treated to date. Unfortunately, the lack of in vivo models reflecting KIT D816V+ advanced disease hampers pathophysiological studies and preclinical development of new therapies for such patients. Here, we describe a new in vivo model of KIT D816V+ advanced systemic mastocytosis developed by transplantation of the human ROSAKIT D816V-Gluc mast cell line in NOD-SCID IL-2R γ-/- mice, using Gaussia princeps luciferase as a reporter. Intravenous injection of ROSAKIT D816V-Gluc cells led, in 4 weeks, to engraftment in all injected primary recipient mice. Engrafted cells were found at high levels in bone marrow, and at lower levels in spleen, liver and peripheral blood. Disease progression was easily monitored by repeated quantification of Gaussia princeps luciferase activity in peripheral blood. This quantification evidenced a linear relationship between the number of cells injected and the neoplastic mast cell burden in mice. Interestingly, the secondary transplantation of ROSAKIT D816V-Gluc cells increased their engraftment capability. To conclude, this new in vivo model mimics at the best the features of human KIT D816V+ advanced systemic mastocytosis. In addition, it is a unique and convenient tool to study the kinetics of the disease and the potential in vivo activity of new drugs targeting neoplastic mast cells.

Human endogenous retrovirus type K (HERV-K) Env protein was previously demonstrated to be overexpressed in human breast cancer (BC) cells and tissues. However, the molecular pathways driving the specific alterations are unknown. We now show that knockdown of its expression with an shRNA (shRNAenv) blocked BC cell proliferation, migration, and invasion. shRNAenv transduction also attenuated the ability of BC cells to form tumors, and notably prevented metastasis. Mechanistically, downregulation of HERV-K blocked expression of tumor-associated genes that included Ras, p-RSK, and p-ERK. The major upstream regulators influenced by HERV-K knockdown were p53, TGF- β1, and MYC. Of interest, when the HERV-K env gene was overexpressed in shRNAenv-transduced BC cells using an HERV-K env expression vector, Ras/Raf/MEK/ERK pathway signaling was restored. CDK5, which alters p53 phosphorylation in some cancers, was upregulated and p53 was downregulated when HERV-K was overexpressed. CDK5 is also a mediator of TGF-β1-induced epithelial-mesenchymal transition and migration in cancer cells, and is involved in tumor formation. Importantly, reductions in migration, invasion, and transformation of BC cells stably transduced with shRNAenv was reversed after adding back a vector with a synonymous mutation of HERV-K env. Taken together, these results indicate that HERV-K Env protein plays an important role in tumorigenesis and metastasis of BC.

PURPOSE: Leukemia stem cells (LSC), which are insensitive to tyrosine kinase inhibitors (TKI), are an important source of TKI resistance and disease relapse in chronic myelogenous leukemia (CML). Obstacles to eradicating LSCs include limited understanding of the regulation network of LSCs. The current study aimed to examine the interplay between NF-κB and FOXM1/β-catenin, and the effect of its chemical intervention on CML LSCs.
EXPERIMENTAL DESIGN: The interplay between NF-κB and FOXM1/β-catenin was analyzed by reciprocal coimmunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP) assay in CML cells. The effect of disturbing NF-κB and FOXM1/β-catenin by niclosamide on the self-renewal capacity and survival of LSCs was evaluated in vitro in human primary CML CD34
RESULTS: Reciprocal co-IP experiments showed physical interaction of p65 and FOXM1. p65 promoted transcription of FOXM1 gene. ChIP assay revealed recruitment of p65 on the promoter of FOXM1 gene. Conversely, FOXM1 and β-catenin positively regulated the nuclear translocation and transcriptional activity of NF-κB in CML cells. Niclosamide disrupted the positive feedback loop between NF-κB and FOXM1/β-catenin, thereby impairing the self-renewal capacity and survival of CML LSCs. Niclosamide decreased the long-term engraftment of human CML LSCs in NOD-SCID IL2Rγ chain-deficient (NOG) mice, and prolonged the survival of CML mice.
CONCLUSIONS: Interaction of p65 with FOXM1/β-catenin is critical in CML and its disruption by niclosamide eradicates LSCs. These findings may improve the understanding of a self-renewal regulatory mechanism of LSCs and offer a rationale-based approach to eliminate LSCs in CML. Clin Cancer Res; 23(3); 789-803. ©2016 AACR.

OBJECTIVE: Our objective was to evaluate the life-long safety profile of gene therapy using retroviral (non-replicating) vectors (nRCR), or cell products in 127 subjects with hemophilia, human immunodeficiency virus (HIV), or cancer, previously treated with such gene therapy.
METHODS: We assessed the occurrence of serious adverse events (SAEs), deaths and presence of replication competent retrovirus (RCR).
RESULTS: A total of 23 subjects remained until the data cut-off date of 31 July 2013 and provided safety information of up to 18 years. Of the 104 subjects who discontinued, the primary reason was loss to follow-up (47.2 %; n = 60). The follow-up period for the 60 subjects lost to follow-up was 7-10 years. A total of 41 subjects experienced at least one SAE, and 15 subjects died. We reviewed SAEs and cause of death (none related to the active therapy), but no evidence was found for safety signals related to new malignancy or neurologic, rheumatological, autoimmune, or hematologic disorder. RCR results were negative, indicating no evidence for in vivo vector persistence.
CONCLUSION: Despite the loss of follow-up, which is the limiting factor in this long-term safety trial, the findings from this long-term follow-up study are encouraging.

In cutaneous T cell lymphomas (CTCL), miR-21 is aberrantly expressed in skin and peripheral blood and displays anti-apoptotic properties in malignant T cells. It is, however, unclear exactly which cells express miR-21 and what mechanisms regulate miR-21. Here, we demonstrate miR-21 expression in situ in both malignant and reactive lymphocytes as well as stromal cells. qRT-PCR analysis of 47 patients with mycosis fungoides (MF) and Sezary Syndrome (SS) confirmed an increased miR-21 expression that correlated with progressive disease. In cultured malignant T cells miR-21 expression was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS T cells and cultured cytokine-dependent SS cells (SeAx). siRNA-mediated depletion of STAT5 inhibited constitutive- and IL-2-induced miR-21 expression in cytokine-independent and dependent T cell lines, respectively. IL-15 and IL-2 were more potent than IL-21 in inducing miR-21 expression in the cytokine-dependent T cells. In conclusion, we provide first evidence that miR-21 is expressed in situ in CTCL skin lesions, induced by IL-2 and IL-15 cytokines, and is regulated by STAT5 in malignant T cells. Thus, our data provide novel evidence for a pathological role of IL-2Rg cytokines in promoting expression of the oncogenic miR-21 in CTCL.

Cholangiocarcinoma is a highly lethal cancer with limited therapeutic options. Recent genomic analysis of cholangiocarcinoma has revealed the presence of fibroblast growth factor receptor 2 (FGFR2) fusion proteins in up to 13% of intrahepatic cholangiocarcinoma (iCCA). FGFR fusions have been identified as a novel oncogenic and druggable target in a number of cancers. In this study, we established a novel cholangiocarcinoma patient derived xenograft (PDX) mouse model bearing an FGFR2-CCDC6 fusion protein from a metastatic lung nodule of an iCCA patient. Using this PDX model, we confirmed the ability of the FGFR inhibitors, ponatinib, dovitinib and BGJ398, to modulate FGFR signaling, inhibit cell proliferation and induce cell apoptosis in cholangiocarcinoma tumors harboring FGFR2 fusions. In addition, BGJ398 appeared to be superior in potency to ponatinib and dovitinib in this model. Our findings provide a strong rationale for the investigation of FGFR inhibitors, particularly BGJ398, as a therapeutic option for cholangiocarcinoma patients harboring FGFR2 fusions.

Prevention of central nervous system (CNS) relapse is critical for cure of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite this, mechanisms of CNS infiltration are poorly understood, and the timing, frequency, and properties of BCP-ALL blasts entering the CNS compartment are unknown. We investigated the CNS-engrafting potential of BCP-ALL cells xenotransplanted into immunodeficient NOD.Cg- ITALIC! Prkdc (ITALIC! scid) ITALIC! Il2rg (ITALIC! tm1Wjl)/SzJ mice. CNS engraftment was seen in 23 of 29 diagnostic samples (79%): 2 of 2 from patients with overt CNS disease and 21 of 27 from patients thought to be CNS negative by diagnostic lumbar puncture. Histologic findings mimic human pathology and demonstrate that leukemic cells transit the blood-cerebrospinal fluid barrier situated close to the dural sinuses, the site of recently discovered CNS lymphatics. Retrieval of blasts from the CNS showed no evidence for chemokine receptor-mediated selective trafficking. The high frequency of infiltration and lack of selective trafficking led us to postulate that CNS tropism is a generic property of leukemic cells. To test this, we performed serial dilution experiments which showed CNS engraftment in 5 of 6 mice after transplant of as few as 10 leukemic cells. Clonal tracking techniques confirmed the polyclonal nature of CNS-infiltrating cells, with multiple clones engrafting in both the CNS and periphery. Overall, these findings suggest that subclinical seeding of the CNS is likely to be present in most BCP-ALL patients at original diagnosis, and efforts to prevent CNS relapse should concentrate on effective eradication of disease from this site rather than targeting entry mechanisms.

Helicobacter pylori (H. pylori) is a spiral-shaped Gram-negative bacterium that causes the most common chronic infection in the human stomach. Approximately 1%-3% of infected individuals develop gastric cancer. However, the mechanisms by which H. pylori induces gastric cancer are not completely understood. The available evidence indicates a strong link between the virulence factor of H. pylori, cytotoxin-associated gene A (CagA), and gastric cancer. To further characterize H. pylori virulence, we established three cell lines by infecting the gastric cancer cell lines SGC-7901 and AGS with cagA+ H. pylori and transfecting SGC-7901 with a vector carrying the full-length cagA gene. We detected 135 differently expressed proteins from the three cell lines using proteome technology, and 10 differential proteins common to the three cell lines were selected and identified by LC-MS/MS as well as verified by western blot: β-actin, L-lactate dehydrogenase (LDH), dihydrolipoamide dehydrogenase (DLD), pre-mRNA-processing factor 19 homolog (PRPF19), ATP synthase, calmodulin (CaM), p64 CLCP, Ran-specific GTPase-activating protein (RanGAP), P43 and calreticulin. Detection of the expression of these proteins and genes encoding these proteins in human gastric cancer tissues by real-time PCR (RT-qPCR) and western blot revealed that the expression of β-ACTIN, LDH, DLD, PRPF19 and CaM genes were up-regulated and RanGAP was down-regulated in gastric cancer tissues and/or metastatic lymph nodes compared to peri-cancerous tissues. High gene expression was observed for H. pylori infection in gastric cancer tissues. Furthermore, the LDH, DLD and CaM genes were demethylated at the promoter -2325, -1885 and -276 sites, respectively, and the RanGAP gene was highly methylated at the promoter -570 and -170 sites in H. pylori-infected and cagA-overexpressing cells. These results provide new insights into the molecular pathogenesis and treatment targets for gastric cancer with H. pylori infection.

Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis.

RNA-binding proteins (RBPs) are increasingly identified as post-transcriptional drivers of cancer progression. The RBP LARP1 is an mRNA stability regulator, and elevated expression of the protein in hepatocellular and lung cancers is correlated with adverse prognosis. LARP1 associates with an mRNA interactome that is enriched for oncogenic transcripts. Here we explore the role of LARP1 in epithelial ovarian cancer, a disease characterized by the rapid acquisition of resistance to chemotherapy through the induction of pro-survival signalling. We show, using ovarian cell lines and xenografts, that LARP1 is required for cancer cell survival and chemotherapy resistance. LARP1 promotes tumour formation in vivo and maintains cancer stem cell-like populations. Using transcriptomic analysis following LARP1 knockdown, cross-referenced against the LARP1 interactome, we identify BCL2 and BIK as LARP1 mRNA targets. We demonstrate that, through an interaction with the 3' untranslated regions (3' UTRs) of BCL2 and BIK, LARP1 stabilizes BCL2 but destabilizes BIK with the net effect of resisting apoptosis. Together, our data indicate that by differentially regulating the stability of a selection of mRNAs, LARP1 promotes ovarian cancer progression and chemotherapy resistance.

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

In addition to the canonical c-Myc p64 and p67 proteins, the human c-myc locus encodes two distinct proteins, mrtl (myc-related translation/localization regulatory factor) and MycHex1 (Myc Human Exon 1), from the upstream open reading frames within the 5'-untranslated region of the c-myc P0 mRNA. The aim of this study is to examine simultaneously, for the first time, mrtl, MycHex1, c-Myc p64, and p67 in human tumor cell lines and pediatric brain tumor tissues. Western blot analysis demonstrated endogenous mrtl, MycHex1, c-Myc p64, and p67 simultaneously. The relative abundance of mrtl and MycHex1 were consistent among a variety of human tumor cell lines, and the relative intensities of mrtl and MycHex1 correlated positively. Confocal imaging revealed mrtl predominantly localized to the nuclear envelope, along with prominent reticular pattern in the cytoplasm. MycHex1 was observed as a series of bright foci located within the nucleus, a subset of which colocalized with fibrillarin. mrtl and MycHex1 co-immunoprecipitated with RACK1, c-Myc, fibrillarin, coilin, and with each other. These findings suggest that mrtl and MycHex1 have multiple interaction partners in both the nucleus and cytoplasm. Sequence analyses confirmed a known polymorphism of mrtl at base 1965 (G>T) and new mutations at bases 1900 (C>G) and 1798 (C>G). Evidence is presented for expression and stable accumulation of all four proteins encoded by three distinct non-overlapping open reading frames within the human c-myc locus. Additional work is warranted to further elucidate the functional or regulatory roles of these molecules in regulation of c-Myc and in oncogenesis.

As rapidly developing patient-derived xenografts (PDX) could represent potential sources of cancer stem cells (CSC), we selected and characterized non-cultured PDX cell suspensions from four different renal carcinomas (RCC). Only the cell suspensions from the serial xenografts (PDX-1 and PDX-2) of an undifferentiated RCC (RCC-41) adapted to the selective CSC medium. The cell suspension derived from the original tumor specimen (RCC-41-P-0) did not adapt to the selective medium and strongly expressed CSC-like markers (CD133 and CD105) together with the non-CSC tumor marker E-cadherin. In comparison, PDX-1 and PDX-2 cells exhibited evolution in their phenotype since PDX-1 cells were CD133high/CD105-/Ecadlow and PDX-2 cells were CD133low/CD105-/Ecad-. Both PDX subsets expressed additional stem cell markers (CD146/CD29/OCT4/NANOG/Nestin) but still contained non-CSC tumor cells. Therefore, using different cell sorting strategies, we characterized 3 different putative CSC subsets (RCC-41-PDX-1/CD132+, RCC-41-PDX-2/CD133-/EpCAMlow and RCC-41-PDX-2/CD133+/EpCAMbright). In addition, transcriptomic analysis showed that RCC-41-PDX-2/CD133- over-expressed the pluripotency gene ERBB4, while RCC-41-PDX-2/CD133+ over-expressed several tumor suppressor genes. These three CSC subsets displayed ALDH activity, formed serial spheroids and developed serial tumors in SCID mice, although RCC-41-PDX-1/CD132+ and RCC-41-PDX-2/CD133+ displayed less efficiently the above CSC properties. RCC-41-PDX-1/CD132+ tumors showed vessels of human origin with CSC displaying peri-vascular distribution. By contrast, RCC-41-PDX-2 originated tumors exhibiting only vessels of mouse origin without CSC peri-vascular distribution.Altogether, our results indicate that PDX murine microenvironment promotes a continuous redesign of CSC phenotype, unmasking CSC subsets potentially present in a single RCC or generating ex novo different CSC-like subsets.