Christina Jamieson

Identification of Process-Localized mRNAs from Cultured Rodent Hippocampal Neurons

The regulated translation of localized mRNAs in neurons provides a mechanism for spatially restricting gene expression in a synapse-specific manner. To identify the population of mRNAs present in distal neuronal processes of rodent hippocampal neurons, we grew neurons on polycarbonate filters etched with 3 μm pores. Although the neuronal cell bodies remained on the top surface of the filters, dendrites, axons, and glial processes penetrated through the pores to grow along the bottom surface of the membrane where they could be mechanically separated from cell bodies. Quantitative PCR and immunochemical analyses of the process preparation revealed that it was remarkably free of somatic contamination. Microarray analysis of RNA isolated from the processes identified over 100 potentially localized mRNAs. In situ hybridization studies of 19 of these transcripts confirmed that all 19 were present in dendrites, validating the utility of this approach for identifying dendritically localized transcripts. Many of the identified mRNAs encoded components of the translational machinery and several were associated with the RNA-binding protein Staufen. These findings indicate that there is a rich repertoire of mRNAs whose translation can be locally regulated and support the emerging idea that local protein synthesis serves to boost the translational capacity of synapses.

Immunosuppressive plasma cells impede T cell-dependent immunogenic chemotherapy

Cancer-associated genetic alterations induce expression of tumour antigens that can activate CD8+ cytotoxic T cells (CTLs), but the microenvironment of established tumours promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumour-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome, and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Our previous studies revealed that B cells recruited by the chemokine CXCL13 into prostate cancer tumours promote the progression of castrate-resistant prostate cancer by producing lymphotoxin, which activates an IκB kinase α (IKKα)-BMI1 module in prostate cancer stem cells. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic agent that is effective in aggressive prostate cancer. We show that mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumour-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFβ receptor signalling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumours.

A Pan-BCL2 Inhibitor Renders Bone-Marrow-Resident Human Leukemia Stem Cells Sensitive to Tyrosine Kinase Inhibition

Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

Reciprocal regulation of SOCS 1 and SOCS3 enhances resistance to ionizing radiation in glioblastoma multiforme.

Purpose: The expression of suppressors of cytokine signaling 1 (SOCS1) and SOCS3 genes is dysregulated in several solid tumors, causing aberrant activation of cell growth and survival signaling pathways. In this study, we analyzed SOCS1 and SOCS3 gene expression in glioblastoma multiforme (GBM) and studied the role of each protein in GBM cell signaling and radiation resistance. Experimental Design: SOCS1 and SOCS3 gene expression was analyzed in 10 GBM cell lines by reverse transcription-PCR and Western blotting. SOCS3 expression was also studied in 12 primary GBM tissues by immunohistochemistry. The methylation status of the SOCS1 and SOCS3 loci was determined by methylation-specific PCR. Extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) activation in GBM cell lines overexpressing SOCS1 or lacking SOCS3 was determined by phosphorylated-specific Western blotting. Radiation responses in SOCS1-positive and SOCS3-deficient GBM cell lines and fibroblasts from wild-type and SOCS1 or SOCS3 knockout mice were studied in a clonogenic survival assay. Results: All GBM cell lines tested lacked SOCS1 expression, whereas GBM cell lines and primary GBM tumor samples constitutively expressed SOCS3. SOCS1 gene repression was linked to hypermethylation of the SOCS1 genetic locus in GBM cells. Reintroduction of SOCS1 or blocking SOCS3 expression sensitized cells to radiation and decreased the levels of activated ERK MAPKs in GBM cells. Conclusions: SOCS1 and SOCS3 are aberrantly expressed in GBM cell lines and primary tissues. Altered SOCS gene expression leads to increased cell signaling through the ERK-MAPK pathway and may play a role in disease pathogenesis by enhancing GBM radioresistance.

Tumor infiltrating B-cells are increased in prostate cancer tissue

BackgroundThe presence of increased B-cell tumor infiltrating lymphocytes (TILs) was seen in mouse prostate cancer (PCa) but has not been fully documented in human PCa. We, therefore, investigated the density of infiltrating B cells within human PCa utilizing a quantitative computational method.MethodsArchived radical prostatectomy specimens from 53 patients with known clinical outcome and D’Amico risk category were obtained and immunohistochemically (IHC) stained for the B cell marker, CD20. Slides were reviewed by a genitourinary pathologist who manually delineated the tumoral regions of PCa. Slides were digitally scanned and a computer algorithm quantified the area of CD20 stained B-cells as a measure of B cell density within the outlined regions of prostate cancer (intra-tumoral region), versus extra-tumoral prostate tissue. Correlations were analyzed between B-cell density and demographic and clinical variables, including D’Amico risk groups and disease recurrence.ResultsFor the entire cohort, the mean intra-tumoral B cell density was higher (3.22 SE = 0.29) than in the extra-tumoral region of each prostatectomy section (2.24, SE = 0.19) (paired t test; P < 0.001). When analyzed according to D’Amico risk group, the intra-tumoral B cell infiltration in low risk (0.0377 vs. 0.0246; p = 0.151) and intermediate risk (0.0260 vs. 0.0214; p = 0.579) patient prostatectomy specimens did not show significantly more B-cells within the PCa tumor. However, patient specimens from the high-risk group (0.0301 vs. 0.0197; p < 0.001) and from those who eventually had PCa recurrence or progression (0.0343 vs. 0.0246; p = 0.019) did show significantly more intra-tumoral CD20+ B-cell staining. Extent of B-cell infiltration in the prostatectomy specimens did not correlate with any other clinical parameters.ConclusionsOur study shows that higher B-cell infiltration was present within the intra-tumoral PCa regions compared to the extra-tumoral benign prostate tissue regions in prostatectomy sections. For this study we developed a new method to measure B-cells using computer-assisted digitized image analysis. Accurate, consistent quantitation of B-cells in prostatectomy specimens is essential for future clinical trials evaluating the effect of B cell ablating antibodies. The interaction of B-cells and PCa may serve as the basis for new therapeutic targets.

Tissue injury and hypoxia promote malignant progression of prostate cancer by inducing CXCL13 expression in tumor myofibroblasts

Significance Prostate cancer often responds to hormone ablation therapy or chemotherapy by becoming more aggressive and metastatic. B cells recruited into hormone-deprived tumors by C-X-C motif chemokine 13 (CXCL13) play an important role in this process. We investigated how androgen ablation induces CXCL13 expression and found that CXCL13 is expressed by myofibroblasts within the tumor microenvironment that become activated as a result of low oxygen tension and hypoxia in androgen-deprived tumors. Hypoxia activates hypoxia-inducible factor 1 (HIF-1) and induces TGF-β expression, which converts fibroblasts to myofibroblasts and stimulates CXCL13 production. We show that several treatments that block CXCL13 expression, including immunodepletion of myofibroblasts, blockade of TGF-β signaling, and phosphodiesterase-5 (PDE5) inhibitors, inhibit B-cell recruitment into androgen-deprived prostate tumors and prevent the emergence of a more aggressive type of cancer. Prostate cancer (PC) is a slowly progressing malignancy that often responds to androgen ablation or chemotherapy by becoming more aggressive, acquiring a neuroendocrine phenotype, and undergoing metastatic spread. We found that B lymphocytes recruited into regressing androgen-deprived tumors by C-X-C motif chemokine 13 (CXCL13), a chemokine whose expression correlates with clinical severity, play an important role in malignant progression and metastatic dissemination of PC. We now describe how androgen ablation induces CXCL13 expression. In both allografted and spontaneous mouse PC, CXCL13 is expressed by tumor-associated myofibroblasts that are activated on androgen ablation through a hypoxia-dependent mechanism. The same cells produce CXCL13 after chemotherapy. Myofibroblast activation and CXCL13 expression also occur in the normal prostate after androgen deprivation, and CXCL13 is expressed by myofibroblasts in human PC. Hypoxia activates hypoxia-inducible factor 1 (HIF-1) and induces autocrine TGF-β signaling that promotes myofibroblast activation and CXCL13 induction. In addition to TGF-β receptor kinase inhibitors, myofibroblast activation and CXCL13 induction are blocked by phosphodiesterase 5 (PDE5) inhibitors. Both inhibitor types and myofibroblast immunodepletion block the emergence of castration-resistant PC in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous metastatic PC with neuroendocrine differentiation.

Dexamethasone induces dysferlin in myoblasts and enhances their myogenic differentiation

Glucocorticoids are beneficial in many muscular dystrophies but they are ineffective in treating dysferlinopathy, a rare muscular dystrophy caused by loss of dysferlin. We sought to understand the molecular basis for this disparity by studying the effects of a glucocorticoid on differentiation of the myoblast cell line, C2C12, and dysferlin-deficient C2C12s. We found that pharmacologic doses of dexamethasone enhanced the myogenic fusion efficiency of C2C12s and increased the induction of dysferlin, along with specific myogenic transcription factors, sarcolemmal and structural proteins. In contrast, the dysferlin-deficient C2C12 cell line demonstrated a reduction in long myotubes and early induction of particular muscle differentiation proteins, most notably, myosin heavy chain. Dexamethasone partially reversed the defect in myogenic fusion in the dysferlin-deficient C2C12 cells. We hypothesize that a key therapeutic benefit of glucocorticoids may be the up-regulation of dysferlin as an important component of glucocorticoid-enhanced myogenic differentiation.

Inhibition of the phosphatidylinositol 3-kinase-Akt pathway enhances gamma-2 herpesvirus lytic replication and facilitates reactivation from latency.

Cellular signalling pathways are critical in regulating the balance between latency and lytic replication of herpesviruses. Here, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway on replication of two gamma-2 herpesviruses, murine gammaherpesvirus-68 (MHV-68) and human herpesvirus-8/Kaposis sarcoma-associated herpesvirus (HHV-8/KSHV). We found that de novo infection of MHV-68 induced PI3K-dependent Akt activation and the lytic replication of MHV-68 was enhanced by inhibiting the PI3K-Akt pathway with both chemical inhibitors and RNA interference technology. Inhibiting the activity of Akt using Akt inhibitor VIII also facilitated the reactivation of KSHV from latency. Both lytic replication and latency depend on the activity of viral transactivator RTA and we further show that the activity of RTA is increased by reducing Akt1 expression. The data suggest that the PI3K-Akt pathway suppresses the activity of RTA and thereby contributes to the maintenance of viral latency and promotes tumorigenesis.

NOTCH1 Signaling Promotes Human T-Cell Acute Lymphoblastic Leukemia Initiating Cell Regeneration in Supportive Niches

Background Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated. Methodology and Principal Findings We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34+ cells from NOTCH1Mutated T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1Wild-type CD34+ cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1Mutated CD34+ fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1Mutated T-ALL LIC-engrafted mice and resulted in depletion of CD34+CD2+CD7+ cells that harbor serial transplantation capacity. Conclusions These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies.

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.