Search

The importance of the immune microenvironment in ovarian cancer progression, metastasis, and response to therapies has become increasingly clear, especially with the new emphasis on immunotherapies. To leverage the power of patient-derived xenograft (PDX) models within a humanized immune microenvironment, three ovarian cancer PDXs were grown in humanized NBSGW (huNBSGW) mice engrafted with human CD34+ cord blood–derived hematopoietic stem cells. Analysis of cytokine levels in the ascites fluid and identification of infiltrating immune cells in the tumors demonstrated that these humanized PDX (huPDX) established an immune tumor microenvironment similar to what has been reported for patients with ovarian cancer. The lack of human myeloid cell differentiation has been a major setback for humanized mouse models, but our analysis shows that PDX engraftment increases the human myeloid population in the peripheral blood. Analysis of cytokines within the ascites fluid of huPDX revealed high levels of human M-CSF, a key myeloid differentiation factor as well as other elevated cytokines that have previously been identified in ovarian cancer patient ascites fluid including those involved in immune cell differentiation and recruitment. Human tumor-associated macrophages and tumor-infiltrating lymphocytes were detected within the tumors of humanized mice, demonstrating immune cell recruitment to tumors. Comparison of the three huPDX revealed certain differences in cytokine signatures and in the extent of immune cell recruitment. Our studies show that huNBSGW PDX models reconstitute important aspects of the ovarian cancer immune tumor microenvironment, which may recommend these models for preclinical therapeutic trials.Significance:huPDX models are ideal preclinical models for testing novel therapies. They reflect the genetic heterogeneity of the patient population, enhance human myeloid differentiation, and recruit immune cells to the tumor microenvironment.

Levels of human M-CSF detected in patient ascites fluid

Human cytokines with significantly higher levels in PDX ascites samples compared to plasma samples

The importance of the immune microenvironment in ovarian cancer progression, metastasis, and response to therapies has become increasingly clear, especially with the new emphasis on immunotherapies. To leverage the power of patient-derived xenograft (PDX) models within a humanized immune microenvironment, three ovarian cancer PDXs were grown in humanized NBSGW (huNBSGW) mice engrafted with human CD34+ cord blood–derived hematopoietic stem cells. Analysis of cytokine levels in the ascites fluid and identification of infiltrating immune cells in the tumors demonstrated that these humanized PDX (huPDX) established an immune tumor microenvironment similar to what has been reported for patients with ovarian cancer. The lack of human myeloid cell differentiation has been a major setback for humanized mouse models, but our analysis shows that PDX engraftment increases the human myeloid population in the peripheral blood. Analysis of cytokines within the ascites fluid of huPDX revealed high levels of human M-CSF, a key myeloid differentiation factor as well as other elevated cytokines that have previously been identified in ovarian cancer patient ascites fluid including those involved in immune cell differentiation and recruitment. Human tumor-associated macrophages and tumor-infiltrating lymphocytes were detected within the tumors of humanized mice, demonstrating immune cell recruitment to tumors. Comparison of the three huPDX revealed certain differences in cytokine signatures and in the extent of immune cell recruitment. Our studies show that huNBSGW PDX models reconstitute important aspects of the ovarian cancer immune tumor microenvironment, which may recommend these models for preclinical therapeutic trials.Significance:huPDX models are ideal preclinical models for testing novel therapies. They reflect the genetic heterogeneity of the patient population, enhance human myeloid differentiation, and recruit immune cells to the tumor microenvironment.

Gain-of-function mutations in the androgen receptor (AR) are found in prostate cancer and are implicated in the failure of hormone therapy. Most studies have emphasized the ligand-binding domain (LBD) where mutations can create promiscuous receptors, but mutations in the NH2-terminal transactivation domain have also been found. To assess AR alteration as a mechanism of treatment resistance, a mouse model (h/mAR-TRAMP) was used in which the murine AR coding region is replaced by human sequence and prostate cancer initiated by a transgenic oncogene. Mice received either no treatment, androgen depletion by castration, or treatment with antiandrogens, and 20 AR transcripts were sequenced per end-stage tumor. All tumors expressed several mutant alleles, although most mutations were low frequency. Some mutations that occurred multiple times within the population were differentially located dependent on treatment. Mutations in castrated or antiandrogen-treated mice were widely dispersed but with a prominent cluster in the LBD (amino acids 736-771), whereas changes in intact mice centered near the NH2-terminal polymorphic glutamine tract. Functional characterization of selected LBD mutant alleles showed diverse effects on AR activity, with about half of the mutations reducing transactivation in vitro. One receptor, AR-R753Q, behaved in a cell- and promoter-dependent manner, although as a germ-line mutation it causes androgen insensitivity syndrome. This suggests that alleles that are loss of function during development may still activate a subset of AR targets to become gain of function in tumorigenesis. Mutant ARs may thus use multiple mechanisms to evade cancer treatment. (Mol Cancer Res 2008;6(11):1691–701)

Supplementary Tables S1-S2 from Profiling Human Androgen Receptor Mutations Reveals Treatment Effects in a Mouse Model of Prostate Cancer

In ovarian cancer, metastasis is typically confined to the peritoneum. Surgical removal of the primary tumor and macroscopic secondary tumors is a common practice, but more effective strategies are needed to target microscopic spheroids persisting in the peritoneal fluid after debulking surgery. To treat this residual disease, therapeutic agents can be administered by either intravenous or intraperitoneal infusion. Here, we describe the use of a cellular Potts model to compare tumor penetration of two classes of drugs (cisplatin and pertuzumab) when delivered by these two alternative routes. The model considers the primary route when the drug is administered either intravenously or intraperitoneally, as well as the subsequent exchange into the other delivery volume as a secondary route. By accounting for these dynamics, the model revealed that intraperitoneal infusion is the markedly superior route for delivery of both small-molecule and antibody therapies into microscopic, avascular tumors typical of patients with ascites. Small tumors attached to peritoneal organs, with vascularity ranging from 2% to 10%, also show enhanced drug delivery via the intraperitoneal route, even though tumor vessels can act as sinks during the dissemination of small molecules. Furthermore, we assessed the ability of the antibody to enter the tumor by in silico and in vivo methods and suggest that optimization of antibody delivery is an important criterion underlying the efficacy of these and other biologics. The use of both delivery routes may provide the best total coverage of tumors, depending on their size and vascularity. Cancer Res; 76(6); 1320–34. ©2015 AACR.

Further details on model assumptions, statistical analysis, and model parameters. Supplementary Figure 1: Vascular Densities in Bowel and Omentum Metastatic Sites for Individual Patients Do Not Correlate. Supplementary Figure 2: Box Plots of Vascular Density in Patient Tumor Samples. Supplementary Table 1: Concentration Fits for IP and IV Compartments.

Supplementary Figure 2 from Treatment-Dependent Androgen Receptor Mutations in Prostate Cancer Exploit Multiple Mechanisms to Evade Therapy

Supplementary Figure 1 from Treatment-Dependent Androgen Receptor Mutations in Prostate Cancer Exploit Multiple Mechanisms to Evade Therapy

The importance of the immune microenvironment in ovarian cancer progression, metastasis, and response to therapies has become increasingly clear, especially with the new emphasis on immunotherapies. In order to leverage the power of patient-derived xenograft (PDX) models within a humanized immune microenvironment, three ovarian cancer PDX were grown in humanized NBSGW mice engrafted with human CD34+ cord blood-derived hematopoietic stem cells. Analysis of cytokine levels in the ascites fluid and infiltrating immune cells in the tumors demonstrated that these humanized PDX (huPDX) established an immune tumor microenvironment similar to what has been reported for ovarian cancer patients. The lack of human myeloid cell differentiation has been a major setback for humanized mouse models, but our analysis shows that PDX engraftment increases the human myeloid population in the peripheral blood. Analysis of cytokines within the ascites fluid of huPDX revealed high levels of human M-CSF, a key myeloid differentiation factor as well as other elevated cytokines that have previously been identified in ovarian cancer patient ascites fluid including those involved in immune cell differentiation and recruitment. Human tumor-associated macrophages and tumor-infiltrating lymphocytes were detected within the tumors of humanized mice, demonstrating immune cell recruitment to tumors. Comparison of the three huPDX revealed certain differences in cytokine signatures and in the extent of immune cell recruitment. Our studies show that huNBSGW PDX models reconstitute important aspects of the ovarian cancer immune tumor microenvironment making this a superior approach for therapeutic trials.

SUMMARYCrosstalk between disparate membrane receptors is thought to drive oncogenic signaling and allow for therapeutic resistance. EGFR and RON are members of two unique receptor tyrosine kinase (RTK) subfamilies that engage in crosstalk through unknown mechanisms. We combined high resolution imaging with biochemical studies and structural mutants to understand how EGFR and RON communicate. We found that EGF stimulation results in EGFR-dependent RON phosphorylation. Crosstalk is unidirectional, since MSP stimulation of RON does not trigger EGFR phosphorylation. Two-color single particle tracking captured the formation of complexes between RON and EGFR, supporting a role for direct interactions in propagating crosstalk. We further show that RON is a substrate for EGFR kinase, and transactivation of RON requires the formation of a signaling competent EGFR dimer. These results identify critical structural features of EGFR/RON crosstalk and provide new mechanistic insights into therapeutic resistance.

In ovarian cancer, metastasis is typically confined to the peritoneum. Surgical removal of the primary tumor and macroscopic secondary tumors is a common practice, but more effective strategies are needed to target microscopic spheroids persisting in the peritoneal fluid after debulking surgery. To treat this residual disease, therapeutic agents can be administered by either intravenous or intraperitoneal infusion. Here, we describe the use of a cellular Potts model to compare tumor penetration of two classes of drugs (cisplatin and pertuzumab) when delivered by these two alternative routes. The model considers the primary route when the drug is administered either intravenously or intraperitoneally, as well as the subsequent exchange into the other delivery volume as a secondary route. By accounting for these dynamics, the model revealed that intraperitoneal infusion is the markedly superior route for delivery of both small-molecule and antibody therapies into microscopic, avascular tumors typical of patients with ascites. Small tumors attached to peritoneal organs, with vascularity ranging from 2% to 10%, also show enhanced drug delivery via the intraperitoneal route, even though tumor vessels can act as sinks during the dissemination of small molecules. Furthermore, we assessed the ability of the antibody to enter the tumor by in silico and in vivo methods and suggest that optimization of antibody delivery is an important criterion underlying the efficacy of these and other biologics. The use of both delivery routes may provide the best total coverage of tumors, depending on their size and vascularity. Cancer Res; 76(6); 1320–34. ©2015 AACR.

ErbB receptors form homodimers and heterodimers between family members. To model ErbB2/ErbB3 signaling, single-particle tracking data are used to create a simulation space with overlapping receptor domains. Stochastic modeling of receptor dimerization and phosphorylation reveals the complexity of ErbB2-3 interactions., Members of the ErbB family of receptor tyrosine kinases are capable of both homointeractions and heterointeractions. Because each receptor has a unique set of binding sites for downstream signaling partners and differential catalytic activity, subtle shifts in their combinatorial interplay may have a large effect on signaling outcomes. The overexpression and mutation of ErbB family members are common in numerous human cancers and shift the balance of activation within the signaling network. Here we report the development of a spatial stochastic model that addresses the dynamics of ErbB3 homodimerization and heterodimerization with ErbB2. The model is based on experimental measures for diffusion, dimer off-rates, kinase activity, and dephosphorylation. We also report computational analysis of ErbB3 mutations, generating the prediction that activating mutations in the intracellular and extracellular domains may be subdivided into classes with distinct underlying mechanisms. We show experimental evidence for an ErbB3 gain-of-function point mutation located in the C-lobe asymmetric dimerization interface, which shows enhanced phosphorylation at low ligand dose associated with increased kinase activity.

Epidermal growth factor receptor kinase mutations drive oncogenesis, but the molecular mechanism of pathological signal initiation is poorly understood. Using high-resolution microscopy methods, the authors reveal that these kinase mutations induce structural changes in the receptor ectodomain that lead to enhanced, ligand-independent dimerization., Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non–small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization.

Ovarian cancer is the leading cause of death from gynecologic cancers in the United States. Failure may be due to variable expression and/or complex interactions of growth factor receptors in individual tumors. As ErbB3-MET cooperativity is implicated in solid tumor resistance to EGFR/ErbB2 inhibitors, we evaluated expression of MET and all 4 ErbB family members in ovarian cancers. Tissue arrays were prepared from archival formalin-fixed paraffin-embedded tumor samples, including 202 ovarian carcinomas (Stage I-IV) and controls. Of 202 patient samples, only 25% were positive for EGFR and 35% for ErbB2 expression. ErbB3, ErbB4, and MET showed marked expression in 76%, 98%, and 96% of cases. Consistent with high incidence, there was no significant correlation for expression of ErbB3, ErbB4, or MET with outcome. On the basis of their high expression in the majority of cases, inhibitors targeting ErbB3, ErbB4, and/or MET may be broadly applicable as therapeutic agents in this disease.

Often considered to be a “dead” kinase, erbB3 is implicated in escape from erbB-targeted cancer therapies. Here, heregulin stimulation is shown to markedly upregulate kinase activity in erbB3 immunoprecipitates. Intact, activated erbB3 phosphorylates tyrosine sites in an exogenous peptide substrate, and this activity is abolished by mutagenesis of lysine 723 in the catalytic domain. Enhanced erbB3 kinase activity is linked to heterointeractions with catalytically active erbB2, since it is largely blocked in cells pretreated with lapatinib or pertuzumab. erbB2 activation of erbB3 is not dependent on equal surface levels of these receptors, since it occurs even in erbB3-transfected CHO cells with disproportionally small amounts of erbB2. We tested a model in which transient erbB3/erbB2 heterointeractions set the stage for erbB3 homodimers to be signaling competent. erbB3 homo- and heterodimerization events were captured in real time on live cells using single-particle tracking of quantum dot probes bound to ligand or hemagglutinin tags on recombinant receptors.

Although ErbB receptors are often expressed in primary ovarian tumors, targeted therapy against these receptors has been minimally successful. Since therapies typically target EGFR or ErbB2 that are present in 25-35% of cases, variability in receptor expression among patients may mask effects. ErbB3 and ErbB4 are more ubiquitous, but targeting these receptors is less common. The timing of treatment with targeted therapies may also be crucial. Clinical trials test these therapies in recurrent platinum-resistant disease, but they might be more effective at preventing recurrence after surgery by eliminating free-floating cancer spheroids that give rise to new solid tumors. Since cancer spheroids are not routinely characterized in ovarian cancer, we isolated spheroids from the malignant ascites of 22 ovarian cancer patients undergoing cytoreductive surgery and analyzed their ErbB receptor signatures by immunofluorescence. Surprisingly, cancer spheroids from nearly all samples (85%) were positive for ErbB2. This differs markedly from the 35% of primary ovarian cancer tumors that were ErbB2 positive in our previous study. NSG mice injected intraperitoneally with cancer spheroids to model disseminated disease developed a combination of solid tumors and malignant ascites. Spheroids obtained from these patient-derived xenograft (PDX) models were also ErbB2 positive. However, solid tumors from the PDX models showed little to no ErbB2 labeling. This altered receptor expression demonstrates the plasticity of ovarian cancer cells as they transition from free-floating spheroids to solid tumors. In the ErbB receptor family, this change may be ErbB2-specific. Analysis of ErbB3 receptor status found no change between spheroids and solid tumors. One implication of this finding is that ErbB2 targeted therapies may be effective in ovarian cancer cases where patients are optimally debulked with only microscopic residual disease. Because spheroids are ErbB2 positive, while solid tumors tend to be ErbB4 and/or ErbB3 positive, we explored the effect of a pan-ErbB inhibitor, afatinib, on tumor burden in our mouse models. Cells were inoculated into the peritoneal cavity and tumors were allowed to develop before treatment. To measure tumor burden in PDX models, anti-HLA-ABC antibody was conjugated to CF750 near-infrared dye and injected IP 24 hours prior to imaging. Tumor burden of fluorescently labeled tumors was measured on the IVIS Spectrum using fluorescence tomography. A decrease in tumor burden and a reduction in ascites was seen in groups treated with afatinib or paclitaxel alone, but particularly with the combined treatment of afatinib and paclitaxel. Supplementing current first-line treatments with ErbB targeted therapies could potentially destroy chemoresistant cells while limiting increased toxicity. Citation Format: Mara P. Steinkamp, Ashley Remy, Irina Lagutina, Carolyn Y. Muller, Diane S. Lidke, Bridget S. Wilson. ErbB2 receptor levels in patient-derived spheroids vs. solid tumors reveal high ovarian cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2511.

Mutations in the androgen receptor (AR) that enable activation by antiandrogens occur in hormone-refractory prostate cancer, suggesting that mutant ARs are selected by treatment. To validate this hypothesis, we compared AR variants in metastases obtained by rapid autopsy of patients treated with flutamide or bicalutamide, or by excision of lymph node metastases from hormone-naïve patients. AR mutations occurred at low levels in all specimens, reflecting genetic heterogeneity of prostate cancer. Base changes recurring in multiple samples or multiple times per sample were considered putative selected mutations. Of 26 recurring missense mutations, most in the NH2-terminal domain (NTD) occurred in multiple tumors, whereas those in the ligand binding domain (LBD) were case specific. Hormone-naïve tumors had few recurring mutations and none in the LBD. Several AR variants were assessed for mechanisms that might underlie treatment resistance. Selection was evident for the promiscuous receptor AR-V716M, which dominated three metastases from one flutamide-treated patient. For the inactive cytoplasmically restricted splice variant AR23, coexpression with AR enhanced ligand response, supporting a decoy function. A novel NTD mutation, W435L, in a motif involved in intramolecular interaction influenced promoter-selective, cell-dependent transactivation. AR-E255K, mutated in a domain that interacts with an E3 ubiquitin ligase, led to increased protein stability and nuclear localization in the absence of ligand. Thus, treatment with antiandrogens selects for gain-of-function AR mutations with altered stability, promoter preference, or ligand specificity. These processes reveal multiple targets for effective therapies regardless of AR mutation. [Cancer Res 2009;69(10):4434–42]

The androgen receptor (AR) is involved in the initiation and progression of prostate cancer and its transition to androgen independence. Genetic variation in AR may contribute to disease risk and has been studied for a polymorphic N-terminal glutamine (Q) tract that shows population heterogeneity. While the length of this tract is known to affect AR in vitro, association with disease is complicated by genetic and environmental factors that have led to discordant epidemiological findings. To clarify the effect of Q tract polymorphism on prostate cancer, we created mice bearing humanized AR genes (h/mAr) varying in Q tract length. ARs with short Q tracts (12Q), which are transcriptionally more active, induce earlier disease in the transgene-induced TRAMP prostate cancer model than alleles with median (21Q) or long (48Q) tracts. Disease length varies within each genotype, with greater differentiation and AR expression in slower growing tumors. Remarkably, following androgen ablation, Q tract length has effects that are also allele-dependent and in directions opposite to those in hormone intact mice. Differences in AR activity conferred by Q tract length thus appear to direct distinct pathways of androgen-independent as well as androgen-dependent progression, and highlight substantial risk that may be associated with alterations in the androgen axis. This AR allelic series in humanized mice provides an experimental paradigm to dissect the role of AR in prostate cancer initiation and progression, to model response to treatment and to test therapies targeted specifically to the human AR.

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with carcinogenesis. Specific mutations common in non-small cell lung cancer (NSCLC), including EGFR-L858R and EGFR-ΔL747-P753, lead to ligand-independent phosphorylation, however the molecular mechanism by which these mutations in the EGFR kinase domain confer constitutive activity remain unknown. Here, using multiple sub-diffraction-limit imaging modalities, we reveal the altered behavior of NSCLC-associated EGFR mutants within the plasma membrane_including altered receptor dimerization, dynamics, and structure_which collectively dysregulate receptor activity. Using multi-color single particle tracking (SPT) and Hidden Markov Model analysis, EGFR mutants are shown to form stable dimers in the absence of ligand and exhibit a slower mobility that is consistent with receptor signaling. These results were confirmed using two-color, single-molecule super-resolution microscopy (dSTORM) to visualize the spatial distribution of receptors. Receptor clustering was quantified by localization-based cross-correlation analysis to show ligand-induced aggregation of EGFR as well as ligand-independent aggregation of EGFR mutants. Since the receptor ectodomain is known to play a critical role in dimerization, live cell FRET measurements between the EGFR N-terminus and the plasma membrane was used to quantify changes in ectodomain structure. We found that unliganded EGFR mutants are more readily found in the extended conformation, similar to the ligand bound wild type receptor. Therefore, mutation within the kinase domain biases the structural equilibrium of the extracellular domain toward a dimer-competent state.Collectively, these data support a model where oncogenic signaling from NSCLC-associated EGFR mutants is a result of productive dimerization between non-ligand bound receptors. Furthermore, because these mutations are found in the kinase domain, this work introduces the concept that oncogenic EGFR signaling may be controlled in part by a form of “inside-out” signaling.

Thiamine-responsive megaloblastic anaemia (TRMA) syndrome with diabetes and deafness was found in two patients from a Tunisian kindred. The proband was homozygous for a novel mutation, 287delG, in the high-affinity thiamine transporter gene, SLC19A2. We demonstrated that fibroblasts from this patient exhibited defective thiamine transport. These data confirm that the SLC19A2 gene is the high-affinity thiamine carrier and that this novel mutation is responsible for TRMA syndrome.

Thiamine-responsive megaloblastic anemia (TRMA) syndrome (OMIM No. 249270) comprises a distinctive triad of clinical features: megaloblastic anemia with ringed sideroblasts, diabetes mellitus, and progressive sensorineural deafness. The TRMA gene has been mapped and cloned. Designated "SLC19A2" as a member of the solute carrier gene superfamily, this gene is mutated in all TRMA kindreds studied to date. The product of the SLC19A2 gene is a membrane protein which transports thiamine (vitamin B1) with sub-micromolar affinity. Cells from TRMA patients are uniquely sensitive to thiamine depletion to the nanomolar range, while pharmacologic doses of vitamin B1 ameliorate the anemia and diabetes. Here we review the current status of studies aimed at understanding the pathophysiology of this unique transport defect.

Thiamine-responsive megaloblastic anaemia with diabetes and deafness1 (TRMA; MIM 249270) is an autosomal recessive disease thought to be due to a defect in thiamine (vitamin B1) transport2,3. Pharmacological doses of thiamine correct the anaemia, and in some cases improve the diabetes, although progressive sensorineural deafness is irreversible4. Previous studies localized the TRMA gene to a 4-cM region on chromosome 1q23.3 (ref. 5), and fine-mapping has recently narrowed that region further6,7. We have previously demonstrated that fibroblasts from people with TRMA lack high-affinity thiamine transport8. Expression of a gene encoding a known yeast thiamine transporter, THI10 (refs 8,9,10), in TRMA mutant cells prevents apoptotic cell death in thiamine-depleted medium. On the basis of these studies, we hypothesized that a defective thiamine transporter causes TRMA. We undertook a candidate gene approach to identify putative thiamine transporters in the 1q23.3 critical region. Here we present evidence that the gene SLC19A2 (for solute carrier family 19 (thiamine transporter), member 2) encodes the first known mammalian thiamine transporter, which we designate thiamine transporter-1 (THTR-1).

The high affinity IgE receptor, FceRI, serves as the primary immunoreceptor on mast cells and basophils.Crosslinking of IgE-bound FceRI via multivalent antigen initiates Src family kinase mediated phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs), recruitment of the kinase Syk, and propagation of signaling through the scaffolding protein Linker for Activation of T-cells (LAT). While the sequence of these signaling events has been well studied using biochemical techniques, the biophysical mechanisms that regulate FceRI signaling are unclear because protein activation and dynamic protein-protein interactions are difficult to quantify on living cells. Using two-color single molecule imaging in live cells, we quantify how FceRI aggregation influences SYK recruitment and LAT mobility.We imaged the basal surface of RBL-2H3 cells expressing GFP-Syk using TIRF microscopy and upon crosslinking of FceRI observed an increase in the membrane localization of GFP-Syk. The formation of GFP-Syk clusters directly co-localized with crosslinked FceRI and the intensity of these clusters increased linearly with FceRI aggregate size. Interestingly, while ensemble measurements showed that FceRI and GFP-Syk were co-localized over minutes, single molecule imaging revealed that GFP-Syk clusters are in fact maintained through a continuous exchange of transiently bound GFP-Syk. We quantify the residency time of individual GFP-Syk molecules at FceRI aggregates and explore the relationship between FceRI aggregate size and Syk residency time. The observed transient GFP-Syk binding supports the hypothesis that phosphorylated SYK moves from FceRI clusters to bind and activate LAT. To explore this mechanism further, we quantify the influence of FceRI crosslinking on the spatiotemporal distribution of LAT. We have generated a Fluorogen Activating Protein (FAP) -tagged LAT construct and take advantage of the far-red emission of the FAP probe to simultaneously track FAP-LAT mobility with respect to FceRI/SYK patches.

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA-mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER-tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER-Golgi and autolysosome secretory pathways, as well as recycling.

Gain-of-function mutations in the androgen receptor (AR) are found in prostate cancer and are implicated in the failure of hormone therapy. Most studies have emphasized the ligand-binding domain (LBD) where mutations can create promiscuous receptors, but mutations in the NH2-terminal transactivation domain have also been found. To assess AR alteration as a mechanism of treatment resistance, a mouse model (h/mAR-TRAMP) was used in which the murine AR coding region is replaced by human sequence and prostate cancer initiated by a transgenic oncogene. Mice received either no treatment, androgen depletion by castration, or treatment with antiandrogens, and 20 AR transcripts were sequenced per end-stage tumor. All tumors expressed several mutant alleles, although most mutations were low frequency. Some mutations that occurred multiple times within the population were differentially located dependent on treatment. Mutations in castrated or antiandrogen-treated mice were widely dispersed but with a prominent cluster in the LBD (amino acids 736-771), whereas changes in intact mice centered near the NH2-terminal polymorphic glutamine tract. Functional characterization of selected LBD mutant alleles showed diverse effects on AR activity, with about half of the mutations reducing transactivation in vitro. One receptor, AR-R753Q, behaved in a cell- and promoter-dependent manner, although as a germ-line mutation it causes androgen insensitivity syndrome. This suggests that alleles that are loss of function during development may still activate a subset of AR targets to become gain of function in tumorigenesis. Mutant ARs may thus use multiple mechanisms to evade cancer treatment. (Mol Cancer Res 2008;6(11):1691–701)

Fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA) syndrome with diabetes and deafness undergo apoptotic cell death in the absence of supplemental thiamine in their cultures. The basis of megaloblastosis in these patients has not been determined. Here we use the stable [1,2-13C2]glucose isotope-based dynamic metabolic profiling technique to demonstrate that defective high-affinity thiamine transport primarily affects the synthesis of nucleic acid ribose via the nonoxidative branch of the pentose cycle. RNA ribose isolated from TRMA fibroblasts in thiamine-depleted cultures shows a time-dependent decrease in the fraction of ribose derived via transketolase, a thiamine-dependent enzyme in the pentose cycle. The fractional rate of de novo ribose synthesis from glucose is decreased several fold 2 to 4 days after removal of thiamine from the culture medium. No such metabolic changes are observed in wild-type fibroblasts or in TRMA mutant cells in thiamine-containing medium. Fluxes through glycolysis are similar in TRMA versus control fibroblasts in the pentose and TCA cycles. We conclude that reduced nucleic acid production through impaired transketolase catalysis is the underlying biochemical disturbance that likely induces cell cycle arrest or apoptosis in bone marrow cells and leads to the TRMA syndrome in patients with defective high-affinity thiamine transport. (Blood. 2003;102: 3556-3561)

A toddler of Haitian descent presented with an 18-month history of chronic consumption coagulopathy, followed by catastrophic skin necrosis. Protein C deficiency (1% to 3% of control) was noted by functional assay; chromogenic assay and antigen levels were 30% of control. Plasma infusion abrogated the disseminated intravascular coagulation-like state. The authors identified a homozygous mutation, C1432T, resulting in a missense, Arg15Trp, in the gamma-carboxyglutamate domain of the protein. Chronic consumption coagulopathy without purpura fulminans or venous thrombosis is a rare presentation of defective protein C pathway. The result of this mutation is a mixed type I (low antigen) and type II (low function) phenotype.

To the editor: The most common mutation in myeloproliferative neoplasm (MPN) substitutes a valine to phenylalanine at position 617 of JAK2 ( JAK2 V617F).[1][1]Recently, Rinaldi et al reported both a nuclear and cytoplasmic localization of JAK2 in K562 cells.[2][2] A previous report also indicated

Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We hypothesize that thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the transporter further, we cloned the murine Slc19a2 locus and characterized the resulting protein. Murine Slc19a2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans ∼13kb with 6 exons, structurally identical to that of the human homolog. We localized the Slc19a2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of Slc19a2 in HEK293T cells resulted in specific uptake of [ 3 H] thiamine, confirming a thiamine transporter function. Western blot analysis of mouse tissues reveals a wide distribution of Slc19a2 protein. Immunohistochemistry studies indicate that Slc19a2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases which play an important role in a number of physiological responses and have also been identified as a common signaling component in several cancer types. Physiological signaling is initiated by ligand binding which stabilizes EGFR dimers, resulting in auto-phosphorylation of the intracellular kinase domain. Mutation of EGFR is associated with tumor progression due to constitutive activity resulting from several known kinase domain variants. Our previous work has shown that ligand-induced dimerization of wild type EGFR results in decreased receptor mobility, and that this reduction in mobility is dependent on kinase activity. Therefore, we wondered whether ligand-independent activity of EGFR mutants correlates with differences in receptor mobility or dimerization kinetics.Here, we employ multi-color, single particle tracking (SPT) of quantum dots to visualize and quantify the dynamics and interactions of individual receptors within the membrane. Using a novel, high-speed hyperspectral microscope, we are able to track up to eight spectrally distinct quantum dots for visualization and quantification of receptor interactions. From this data, we quantify receptor dimerization kinetics using a Hidden Markov Model. We find that unliganded EGFR mutants exhibit a reduced mobility and longer dimer lifetimes when compared to wild type. These results are consistent with constitutive activity of the EGFR mutants and suggest that the active kinase domain facilitates dimer formation in the absence of ligand. Moreover, we test the effects of clinical tyrosine kinase inhibitors on the single molecule behavior of EGFR mutants. We further examined the mobility and clustering of EGFR within the cellular actin cytoskeleton by hyperspectral tracking over GFP-actin, as well as two-color super-resolution microscopy.

Abstract 2820 Background: Mpl, the receptor for thrombopoietin (Tpo), activates Jak2. Mpl and Jak2 are expressed both on platelets and on hematopoietic progenitors, where they initiate critical cell survival and proliferation signals in response to Tpo. Thus, interfering with the “Mpl/Jak2 couple” is likely to alter myelopoiesis. Indeed mutations in JAK2 or MPL are characteristic of myeloproliferative neoplasms (MPN). However, platelet Mpl expression is frequently low in MPN, including MPN with no mutation of JAK2. Aim: To determine whether expression of wild type Jak2 (Jak2wt) is altered in MPN, and whether changes in expression of Jak2wt affect Mpl expression. Methods: Mpl and Jak2 protein expression was analysed by immunoblotting in platelet and cell lysates. Expression of JAK2 wt and JAK2 V617F was also studied at the mRNA level using quantitative RT-PCR. Transient transfections of HEL and UKE-1 cells were used to study the effects of Jak2wt on Mpl and of MPL on Jak2wt and Jak2V617F. We also studied the cellular localization of Jak2wt, Jak2V617F and Mpl, expressed as chimeric proteins fused to Visible Fluorescent Proteins (VFP, derivatives of GFP) or detected by immunofluorescence methods. Results: Following SDS-PAGE and western blotting of normal platelet proteins, Mpl was typically found in two isoforms: the long, mature 84 kDa isoform (Golgi-processed, glycosylated, functional, Tpo-binding form expressed at the cell surface) and the short, 74 kDa isoform (immature Mpl). Four subgroups of MPN patients were defined according to platelet Mpl patterns: pattern 1, mature and immature Mpl expressed at comparable levels, typical of healthy donors and found in 37% of MPN tested; pattern 2, mature Mpl only, typical of reactive states, associated with high JAK2 wt expression and observed in 15% of MPN; pattern 3: immature Mpl only; and pattern 4: no Mpl. Patterns 3 and 4 were observed in 48% of MPN and were associated with low JAK2 wt levels ( Transfection of JAK2wt in HEL and UKE-1 cells, alone or together with MPL, increased the yield of mature Mpl. Fluorescence studies of HEL cells revealed that both endogenous Mpl and the Mpl-VFP chimeric proteins were largely restricted to an intracellular pool, without significant co-localization with Jak2V617F. In contrast, significant levels of transfected Mpl-VFP reach the plasma membrane in K562 cells that express only Jak2wt; co-localization of Jak2 and Mpl in these cells was significant. Conclusion: JAK2 wt and Mpl levels are linked and both are frequently low in MPN, including JAK2 V617F-negative MPN. Co-localization of Mpl and Jak2V617F is poor whereas Mpl and Jak2wt are strongly co-localized, consistent with constitutive association and indicating a role for Jak2wt as chaperone along the exocytic or recycling pathways. Future work will seek to determine the intracellular location of aberrantly trafficked Mpl and establish if other mutations found in MPN are linked to defects in the expression or trafficking of Mpl. Disclosures: No relevant conflicts of interest to declare.

Abstract 2861 Background: Mpl, the receptor for thrombopoietin (Tpo), activates Jak2. Mpl and Jak2 are expressed both on platelets and on hematopoietic progenitors, where they initiate critical cell survival and proliferation signals in response to Tpo. Thus, interfering with the “Mpl/Jak2 pair” is likely to alter myelopoiesis. Indeed, Bcr-Abl-negative myeloproliferative neoplasms (MPNs) are characterized by the presence of an activating mutation of the JAK2 gene (JAK2V617F) and platelets with low or absent expression of Mpl. However, aberrant Mpl expression (absence of the mature form, accumulation of the ER core-glycosylated form) in MPNs has been linked to low expression levels of wild type JAK2 (JAK2WT). Aim: To determine how the presence of JAK2WT or JAK2V617F impacts Mpl trafficking to the cell surface in MPNs. Methods: Transient transfections of HEL cells (JAK2 V617F+/+) and K562 cells (JAK2 WT+/+ and expressing the Bcr-Abl fusion protein characteristic of chronic myelogenous leukemia) were used to study the effects of Jak2WT and Jak2V617F on Mpl. We also studied the cellular localization of Jak2WT, Jak2V617F and Mpl, detected by immunofluorescence or expressed as chimeric proteins fused to Visible Fluorescent Proteins (VFP, derivatives of GFP). The sub-cellular compartment containing the core-glycosylated (immature) form of Mpl was identified by confocal co-localization studies with specific intracellular markers as well as by using a novel genetically encoded fluorescent protein (MiniSOG) recently developed for correlated light and electron microscopic analyses. Finally, Mpl trafficking was evaluated in HEL and K562 cells using a cell surface biotinylation assay. Results: In HEL cells, re-introduction of Jak2WT, alone or with MPL, favors an increase in the ratio of mature:immature Mpl. Transfected MplmOrange2, in K562 cells, shows a significant co-localization with transfected and endogenous JAK2 WT, but not with transfected JAK2 V617F-mCitrine. In addition, in HEL cells, transfected MplmOrange2 does not show a significant co-localization with endogenous JAK2V617F. Both endogenous Mpl and transfected MplmOrange2 were detected in punctate structures that stain positively with ER-Tracker and ER exit site (ERES) markers. These structures are negative for other common ER markers, as well as Golgi and endosomal proteins. Ultrastructural electron microscopy studies based on expression of MplmKO2-MiniSOG revealed that Mpl accumulates in multivesicular bodies that bud from the ER. Consistent with an autophagic process, Mpl-containing structures stain positive for LC3. Thrombopoietin stimulus results in rapid delivery of ER core-glycosylated Mpl to the surface, likely through the “unconventional” autolysosome-mediated pathway. Conclusion: Co-localization of Mpl and Jak2V617F is poor, whereas Mpl and Jak2WT are strongly co-localized. These results support the role of wild type Jak2 in the stabilization at the surface of Mpl and are indicating a role for Jak2WT as a chaperone for the exocytic or recycling pathways. Furthermore, our data obtained in transfected hematopoietic cells strongly suggest that aberrant Mpl trafficking occurs via a secretory autolysosome pathway. This provides a plausible route for limited amounts of ER-core glycosylated Mpl receptor to reach the cell surface. The emerging field of autophagy-based unconventional secretion, associated with such diverse processes as inflammation and tissue remodeling, may now be associated with both the causation and potential treatment of myeloproliferative diseases. Disclosures: No relevant conflicts of interest to declare.