Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence.

The TGF-ß-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-ß pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.

International Guidelines for Veterinary Tumor Pathology: A Call to Action.

Standardization of tumor assessment lays the foundation for validation of grading systems, permits reproducibility of oncologic studies among investigators, and increases confidence in the significance of study results. Currently, there is minimal methodological standardization for assessing tumors in veterinary medicine, with few attempts to validate published protocols and grading schemes. The current article attempts to address these shortcomings by providing standard guidelines for tumor assessment parameters and protocols for evaluating specific tumor types. More detailed information is available in the Supplemental Files, the intention of which is 2-fold: publication as part of this commentary, but more importantly, these will be available as "living documents" on a website (www.vetcancerprotocols.org), which will be updated as new information is presented in the peer-reviewed literature. Our hope is that veterinary pathologists will agree that this initiative is needed, and will contribute to and utilize this information for routine diagnostic work and oncologic studies. Journal editors and reviewers can utilize checklists to ensure publications include sufficient detail and standardized methods of tumor assessment. To maintain the relevance of the guidelines and protocols, it is critical that the information is periodically updated and revised as new studies are published and validated with the intent of providing a repository of this information. Our hope is that this initiative (a continuation of efforts published in this journal in 2011) will facilitate collaboration and reproducibility between pathologists and institutions, increase case numbers, and strengthen clinical research findings, thus ensuring continued progress in veterinary oncologic pathology and improving patient care.

Inter-Institutional Partnerships to Develop Veterinarian-Investigators through the NIH Comparative Biomedical Scientist Training Program Benefit One Health Goals.

Limitations in workforce size and access to resources remain perennial challenges to greater progress in academic veterinary medicine and engagement between human and veterinary medicine (One Health). Ongoing resource constraints occur in part due to limited public understanding of the role veterinarians play in improving human health. One Health interactions, particularly through interdisciplinary collaborations in biomedical research, present constructive opportunities to inform resource policies and advance health care. To this end, inter-institutional partnerships between individual veterinary medical education programs (VMEPs) and several National Institutes of Health (NIH) intramural research programs have created synergies beyond those provided by individual programs. In the NIH Comparative Biomedical Scientist Training Program (CBSTP), interdisciplinary cross-training of veterinarians consisting of specialty veterinary medicine coupled with training in human disease research leading to a PhD, occurs collaboratively on both VMEP and NIH campuses. Pre-doctoral veterinary student research opportunities have also been made available. Through the CBSTP, NIH investigators and national biomedical science policy makers gain access to veterinary perspective and expertise, while veterinarians obtain additional opportunities for NIH-funded research training. CBSTP Fellows serve as de facto ambassadors enhancing visibility for the profession while in residence at NIH, and subsequently through a variety of university, industry, and government research appointments, as graduates. Thus, the CBSTP represents an inter-institutional opportunity that not only addresses critical needs for veterinarian-scientists in the biomedical workforce, but also simultaneously exposes national policy makers to veterinarian-scientists' specialized training, leading to more effective realization of One Health goals to benefit human and animal health.

Oncogenesis driven by the Ras/Raf pathway requires the SUMO E2 ligase Ubc9.

The small GTPase KRAS is frequently mutated in human cancer and currently there are no targeted therapies for KRAS mutant tumors. Here, we show that the small ubiquitin-like modifier (SUMO) pathway is required for KRAS-driven transformation. RNAi depletion of the SUMO E2 ligase Ubc9 suppresses 3D growth of KRAS mutant colorectal cancer cells in vitro and attenuates tumor growth in vivo. In KRAS mutant cells, a subset of proteins exhibit elevated levels of SUMOylation. Among these proteins, KAP1, CHD1, and EIF3L collectively support anchorage-independent growth, and the SUMOylation of KAP1 is necessary for its activity in this context. Thus, the SUMO pathway critically contributes to the transformed phenotype of KRAS mutant cells and Ubc9 presents a potential target for the treatment of KRAS mutant colorectal cancer.

Naturally Occurring Canine Melanoma as a Predictive Comparative Oncology Model for Human Mucosal and Other Triple Wild-Type Melanomas.

Melanoma remains mostly an untreatable fatal disease despite advances in decoding cancer genomics and developing new therapeutic modalities. Progress in patient care would benefit from additional predictive models germane for human disease mechanisms, tumor heterogeneity, and therapeutic responses. Toward this aim, this review documents comparative aspects of human and naturally occurring canine melanomas. Clinical presentation, pathology, therapies, and genetic alterations are highlighted in the context of current basic and translational research in comparative oncology. Somewhat distinct from sun exposure-related human cutaneous melanomas, there is growing evidence that a variety of gene copy number alterations and protein structure/function mutations play roles in canine melanomas, in circumstances more analogous to human mucosal melanomas and to some extent other melanomas with murine sarcoma viral oncogene homolog B (BRAF), Neuroblastoma RAS Viral (V-Ras) Oncogene Homolog (NRAS), and neurofibromin 1 tumor suppressor NF1 triple wild-type genotype. Gaps in canine genome annotation, as well as an insufficient number and depth of sequences covered, remain considerable barriers to progress and should be collectively addressed. Preclinical approaches can be designed to include canine clinical trials addressing immune modulation as well as combined-targeted inhibition of Rat Sarcoma Superfamily/Mitogen-activated protein kinase (RAS/MAPK) and/or Phosphatidylinositol-3-Kinase/Protein Kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal transduction, pathways frequently activated in both human and canine melanomas. Future investment should be aimed towards improving understanding of canine melanoma as a predictive preclinical surrogate for human melanoma and for mutually benefiting these uniquely co-dependent species.

Bioluminescent imaging of drug efflux at the blood-brain barrier mediated by the transporter ABCG2.

ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood-brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that D-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter.

B cell-specific deficiencies in mTOR limit humoral immune responses.

Generation of high-affinity Abs in response to Ags/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Ab responses to Ag stimulation require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either the model Ag 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken γ globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to form germinal centers, develop high-affinity anti-NP-specific or anti-Pn14-specific Abs, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates and had lower pneumococcal surface protein A-specific Ab titers when immunized and challenged with live S. pneumoniae infection. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity Ab responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR KI and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B cell-specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in WT B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity Abs.

Detection of differential fetal and adult expression of chloride intracellular channel 4 (CLIC4) protein by analysis of a green fluorescent protein knock-in mouse line.

BACKGROUND: Chloride Intracellular Channel 4 (CLIC4) is one of seven members in the closely related CLIC protein family. CLIC4 is involved in multiple cellular processes including apoptosis, cellular differentiation, inflammation and endothelial tubulogenesis. Despite over a decade of research, no comprehensive in situ expression analysis of CLIC4 in a living organism has been reported. In order to fulfill this goal, we generated a knock-in mouse to express Green Fluorescent Protein (GFP) from the CLIC4 locus, thus substituting the GFP coding region for CLIC4. We used GFP protein expression to eliminate cross reaction with other CLIC family members. RESULTS: We analyzed CLIC4 expression during embryonic development and adult organs. During mid and late gestation, CLIC4 expression is modulated particularly in fetal brain, heart, thymus, liver and kidney as well as in developing brown adipose tissue and stratifying epidermis. In the adult mouse, CLIC4 is highly expressed globally in vascular endothelial cells as well as in liver, lung alveolar septae, pancreatic acini, spermatogonia, renal proximal tubules, cardiomyocytes and thymic epithelial cells. Neural expression included axonal tracks, olfactory bulb, Purkinje cell layer and dentate gyrus. Renal CLIC4 expression was most pronounced in proximal tubules, although altered renal function was not detected in the absence of CLIC4. Myeloid cells and B cells of the spleen are rich in CLIC4 expression as are CD4 and CD8 positive T cells. CONCLUSIONS: In a comprehensive study detailing CLIC4 expression in situ in a mouse model that excludes cross reaction with other family members, we were able to document previously unreported expression for CLIC4 in developing fetus, particularly the brain. In addition, compartmentalized expression of CLIC4 in specific adult tissues and cells provides a focus to explore potential functions of this protein not addressed previously.

Normal viability of Kai1/Cd82 deficient mice.

The KAI1/CD82 tetraspanin is a widely expressed cell surface molecule thought to organize diverse cellular signaling processes. KAI1/CD82 suppresses metastasis but not tumorigenicity, establishing it as one of a class of metastasis suppressor genes. In order to further assess its functions, we have characterized the phenotypic properties of Kai1/Cd82 deleted mice, including viability, fertility, lymphocyte composition, blood chemistry and tissue histopathology, and of their wild-type and heterozygote littermates. Interestingly, Kai1/Cd82(-/-) showed no obvious genotype associated defects in any of these processes and displayed no genotype associated histopathologic abnormalities after 12 or 18 months of life. Expression profiles of non-immortal, wild-type and Kai1/Cd82(-/-) mouse embryo fibroblast (MEFs) indicated distinct sex-specific and genotype-specific profiles. These data identify 191 and 1,271 differentially expressed transcripts (by twofold at P < 0.01) based on Kai1/CD82 genotype status in female and male MEFs, respectively. Differentially expressed genes in male MEFs were surprisingly enriched for cell division related processes, suggesting that Kai1/Cd82 may functionally affect these processes. This suggests that Kai/Cd82 has an unappreciated role in the early establishment of proliferation and division when challenged with a new environment that might play a role in adaptability to new metastatic sites.

Augmented IL-15Rα expression by CD40 activation is critical in synergistic CD8 T cell-mediated antitumor activity of anti-CD40 antibody with IL-15 in TRAMP-C2 tumors in mice.

IL-15 has potential as an immunotherapeutic agent for cancer treatment because it is a critical factor for the proliferation and activation of NK and CD8(+) T cells. However, monotherapy of patients with malignancy with IL-15 that has been initiated may not be optimal, because of the limited expression of the private receptor, IL-15Rα. We demonstrated greater CD8 T cell-mediated therapeutic efficacy using a combination regimen of murine IL-15 administered with an agonistic anti-CD40 Ab (FGK4.5) that led to increased IL-15Rα expression on dendritic cells (DCs), as well as other cell types, in a syngeneic established TRAMP-C2 tumor model. Seventy to one hundred percent of TRAMP-C2 tumor-bearing wild-type C57BL/6 mice in the combination group manifested sustained remissions, whereas only 0-30% in the anti-CD40-alone group and none in the murine IL-15-alone group became tumor free (p < 0.001). However, the combination regimen showed less efficacy in TRAMP-C2 tumor-bearing IL-15Rα(-/-) mice than in wild-type mice. The combination regimen significantly increased the numbers of TRAMP-C2 tumor-specific SPAS-1/SNC9-H(8) tetramer(+)CD8(+) T cells, which were associated with the protection from tumor development on rechallenge with TRAMP-C2 tumor cells. Using an in vitro cytolytic assay that involved NK cells primed by wild-type or IL-15Rα(-/-) bone marrow-derived DCs, we demonstrated that the expression of IL-15Rα by DCs appeared to be required for optimal IL-15-induced NK priming and killing. These findings support the view that anti-CD40-mediated augmented IL-15Rα expression was critical in IL-15-associated sustained remissions observed in TRAMP-C2 tumor-bearing mice receiving combination therapy.

Tongue orthotopic xenografts to study fusion-negative rhabdomyosarcoma invasion and metastasis in live animals.

PAX3/7 fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Limited understanding of advanced FN-RMS is partially attributed to the absence of sequential invasion and dissemination events and the challenge in studying cell behavior, using, for example, non-invasive intravital microscopy (IVM), in currently used xenograft models. Here, we developed an orthotopic tongue xenograft model of FN-RMS to study cell behavior and the molecular basis of invasion and metastasis using IVM. FN-RMS cells are retained in the tongue and invade locally into muscle mysial spaces and vascular lumen, with evidence of hematogenous dissemination to the lungs and lymphatic dissemination to lymph nodes. Using IVM of tongue xenografts reveals shifts in cellular phenotype, migration to blood and lymphatic vessels, and lymphatic intravasation. Insight from this model into tumor invasion and metastasis at the tissue, cellular, and subcellular level can guide new therapeutic avenues for advanced FN-RMS.

Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression.

Control of organ size by cell proliferation and survival is a fundamental developmental process, and its deregulation leads to cancer. However, the molecular mechanism underlying organ size control remains elusive in vertebrates. In Drosophila, the Hippo (Hpo) signaling pathway controls organ size by both restricting cell growth and proliferation and promoting cell death. Here we investigated whether mammals also require the Hpo pathway to control organ size and adult tissue homeostasis. We found that Mst1 and Mst2, the two mouse homologs of the Drosophila Hpo, control the sizes of some, but not all organs, in mice, and Mst1 and Mst2 act as tumor suppressors by restricting cell proliferation and survival. We show that Mst1 and Mst2 play redundant roles, and removal of both resulted in early lethality in mouse embryos. Importantly, tumors developed in the liver with a substantial increase of the stem/progenitor cells by 6 months after removing Mst1 and Mst2 postnatally. We show that Mst1 and Mst2 were required in vivo to control Yap phosphorylation and activity. Interestingly, apoptosis induced by TNFalpha was blocked in the Mst1 and Mst2 double-mutant cells both in vivo and in vitro. As TNFalpha is a pleiotropic inflammatory cytokine affecting most organs by regulating cell proliferation and cell death, resistance to TNFalpha-induced cell death may also contribute significantly to tumor formation in the absence of Mst1 and Mst2.

Fusion-negative rhabdomyosarcoma orthotopic tongue xenografts for study of invasion, intravasation and metastasis in live animals.

PAX3/7 Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Towards achieving a more complete understanding of advanced FN-RMS, we developed an orthotopic tongue xenograft model for studies of molecular basis of FN-RMS invasion and metastasis. The behavior of FN-RMS cells injected into murine tongue was examined using in vivo bioluminescence imaging, non-invasive intravital microscopy (IVM), and histopathology and compared to the prevailing hindlimb intramuscular and subcutaneous xenografts. FN-RMS cells were retained in the tongue and invaded locally into muscle mysial spaces and vascular lumen. While evidence of hematogenous dissemination to the lungs occurred in tongue and intramuscular xenografts, evidence of local invasion and lymphatic dissemination to lymph nodes only occurred in tongue xenografts. IVM and RNA-seq of tongue xenografts reveal shifts in cellular phenotype and differentiation state in tongue xenografts. IVM also shows homing to blood and lymphatic vessels, lymphatic intravasation, and dynamic membrane protrusions. Based on these findings, the tongue orthotopic xenograft of FN-RMS is a valuable model for tumor progression studies at the tissue, cellular and subcellular levels providing insight into kinetics and molecular bases of tumor invasion and metastasis and, hence, new therapeutic avenues for advanced FN-RMS.

Drug combinations identified by high-throughput screening promote cell cycle transition and upregulate Smad pathways in myeloma.

Drug resistance and disease progression are common in multiple myeloma (MM) patients, underscoring the need for new therapeutic combinations. A high-throughput drug screen in 47 MM cell lines and in silico Huber robust regression analysis of drug responses revealed 43 potentially synergistic combinations. We hypothesized that effective combinations would reduce MYC expression and enhance p16 activity. Six combinations cooperatively reduced MYC protein, frequently over-expressed in MM and also cooperatively increased p16 expression, frequently downregulated in MM. Synergistic reductions in viability were observed with top combinations in proteasome inhibitor-resistant and sensitive MM cell lines, while sparing fibroblasts. Three combinations significantly prolonged survival in a transplantable Ras-driven allograft model of advanced MM closely recapitulating high-risk/refractory myeloma in humans and reduced viability of ex vivo treated patient cells. Common genetic pathways similarly downregulated by these combinations promoted cell cycle transition, whereas pathways most upregulated were involved in TGFß/SMAD signaling. These preclinical data identify potentially useful drug combinations for evaluation in drug-resistant MM and reveal potential mechanisms of combined drug sensitivity.

Proteomic profiling of H-Ras-G12V induced hypertrophic cardiomyopathy in transgenic mice using comparative LC-MS analysis of thin fresh-frozen tissue sections.

Determination of disease-relevant proteomic profiles from limited tissue specimens, such as pathological biopsies and tissues from small model organisms, remains an analytical challenge and a much needed clinical goal. In this study, a transgenic mouse disease model of cardiac-specific H-Ras-G12V induced hypertrophic cardiomyopathy provided a system to explore the potential of using mass spectrometry (MS)-based proteomics to obtain a disease-relevant molecular profile from amount-limited specimens that are routinely used in pathological diagnosis. Our method employs a two-stage methanol-assisted solubilization to digest lysates prepared from 8-µm-thick fresh-frozen histological tissue sections of diseased/experimental and normal/control hearts. Coupling this approach with a nanoflow reversed-phase liquid chromatography (LC) and a hybrid linear ion trap/Fourier transform-ion cyclotron resonance MS resulted in the identification of 704 and 752 proteins in hypertrophic and wild-type (control) myocardium, respectively. The disease driving H-Ras protein along with vimentin were unambiguously identified by LC-MS in hypertrophic myocardium and cross-validated by immunohistochemistry and western blotting. The pathway analysis involving proteins identified by MS showed strong association of proteomic data with cardiovascular disease. More importantly, the MS identification and subsequent cross-validation of Wnt3a and ß-catenin, in conjunction with IHC identification of phosphorylated GSK-3ß and nuclear localization of ß-catenin, provided evidence of Wnt/ß-catenin canonical pathway activation secondary to Ras activation in the course of pathogenic myocardial hypertrophic transformation. Our method yields results indicating that the described proteomic approach permits molecular discovery and assessment of differentially expressed proteins regulating H-Ras induced hypertrophic cardiomyopathy. Selected proteins and pathways can be further investigated using immunohistochemical techniques applied to serial tissue sections of similar or different origin.

CLIC4 is a tumor suppressor for cutaneous squamous cell cancer.

Chloride intracellular channel (CLIC) 4 is a member of a redox-regulated, metamorphic multifunctional protein family, first characterized as intracellular chloride channels. Current knowledge indicates that CLICs participate in signaling, cytoskeleton integrity and differentiation functions of multiple tissues. In metabolically stressed skin keratinocytes, cytoplasmic CLIC4 is S-nitrosylated and translocates to the nucleus where it enhances transforming growth factor-ß (TGF-ß) signaling by protecting phospho-Smad 2 and 3 from dephosphorylation. CLIC4 expression is diminished in multiple human epithelial cancers, and the protein is excluded from the nucleus. We now show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines, and the protein is excluded from the nucleus. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear translocation of CLIC4. Adenoviral-mediated reconstitution of nuclear CLIC4 in squamous cancer cells enhances TGF-ß-dependent transcriptional activity and inhibits growth. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts inhibits tumor growth, whereas elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor growth and enhances TGF-ß signaling. These results indicate that CLIC4 suppresses the growth of squamous cancers, that reduced CLIC4 expression and nuclear residence detected in cancer cells is associated with the altered redox state of tumor cells and the absence of detectable nuclear CLIC4 in cancers contributes to TGF-ß resistance and enhances tumor development.

Prostate epithelial Pten/TP53 loss leads to transformation of multipotential progenitors and epithelial to mesenchymal transition.

Loss of PTEN and loss of TP53 are common genetic aberrations occurring in prostate cancer. PTEN and TP53 contribute to the regulation of self-renewal and differentiation in prostate progenitors, presumptive tumor initiating cells for prostate cancer. Here we characterize the transformed phenotypes resulting from deletion of the Pten and TP53 tumor suppressors in prostate epithelium. Using the PB-Cre4(+)Pten(fl/fl)TP53(fl/fl) model of prostate cancer, we describe the histological and metastatic properties of primary tumors, transplanted primary tumor cells, and clonal cell lines established from tumors. Adenocarcinoma was the major primary tumor type that developed, which progressed to lethal sarcomatoid carcinoma at approximately 6 months of age. In addition, basal carcinomas and prostatic urothelial carcinomas were observed. We show that tumor heterogeneity resulted, at least in part, from the transformation of multipotential progenitors. CK8+ luminal epithelial cells were capable of undergoing epithelial to mesenchymal transition in vivo to sarcomatoid carcinomas containing osseous metaplasia. Metastasis rarely was observed from primary tumors, but metastasis to lung and lymph nodes occurred frequently from orthotopic tumors initiated from a biphenotypic clonal cell line. Androgen deprivation influenced the differentiated phenotypes of metastases. These data show that one functional consequence of Pten/TP53 loss in prostate epithelium is lineage plasticity of transformed cells.

Genetic evidence for the role of Erk activation in a lymphoproliferative disease of mice.

Germline mutation of the linker for activation of T cells (LAT) gene at the phospholipase C-gamma1 (PLC-gamma1)-binding site leads to a fatal lymphoproliferative disease in mice. The hyperactivated T cells that develop in these mice have defective T-cell antigen receptor (TCR)-induced calcium flux but enhanced mitogen-activated protein kinase (MAPK) activation. We used genetic analysis to investigate genes whose products might suppress MAPK activation and lymphoproliferative disease in LAT mutant mice. B-lymphocyte adaptor molecule of 32 kDa (Bam32) is a known mediator of MAPK activation in B cells. We recently reported that in CD4(+) T cells, Bam32 deficiency decreased MAPK activation and specifically extracellular-signal-regulated kinase (Erk) signaling, following TCR stimulation. By crossing the Bam32 null mutation onto the LAT knock-in background, we found that the Bam32 null mutation delayed the onset and decreased the severity of lymphoproliferative disease in LAT knock-in mice. The pulmonary lymphocyte infiltration seen in LAT knock-in mice was also markedly decreased in double-mutant mice. Additionally, Erk activation was diminished in LAT knock-in Bam32 knockout CD4(+) T cells. To more accurately determine the role of Erk in this delay of lymphoproliferative disease, we also bred a transgenic, hypersensitive Erk allele (the Erk2 sevenmaker mutant) onto the LAT knock-in Bam32 knockout double-mutant background. These triple transgenic mice demonstrated a role for Erk activation in lymphoproliferative disease caused by the LAT knock-in mutation.

Pharmacokinetics and tolerability of the dual TORC1/2 inhibitor sapanisertib in combination with the MEK inhibitor trametinib in dogs.

Activation of one or both the Ras/MAPK and PI3K/Akt/mTOR signal transduction pathways are known to mediate oncogenicity of several canine and human cancers, including mucosal melanomas. Reciprocal cross activation between the two pathways can be a source of drug resistance. Consequently, oral dosing for plasma pharmacokinetic (PK) analysis and tolerability to a combination of sapanisertib, a dual TORC1/2 inhibitor, and trametinib, a MEK inhibitor, was evaluated in nontumor-bearing laboratory dogs for its potential application in parallel pathway targeting. Twelve dogs, divided into three equal cohorts, received either the combination or single agents. Animals were monitored for PK following single dose and 17-day repeat dosing, and by clinical observations, hematology, serum biochemistry, coagulation studies and urinalyses. A single trametinib dose (0.025 mg/kg), sulfated as dimethyl sulfoxide which enhanced its absorption, reached mean maximum concentration (Cmax) 0.64 ng/mL [18% coefficient of variation (CV)] at a median time to maximum concentration (Tmax) of 1.5 h (hr), and mean area under the concentration-time curve (AUC) 16.8 hr*ng/mL (14%CV), which were similar when given alone or in combination with sapanisertib. A prolonged half-life afforded 3-4-fold plasma accumulation of trametinib with daily dosing, analogous to humans. Trametinib PK mirrored previous regulatory data in dogs, while exposure approximated some published human values but generally not all patients. Sapanisertib-alone in canine plasma following single 0.1 mg/kg dose [mean Cmax 26.3 ng/mL (21%CV), median Tmax 2.0 hr, and mean AUC 248 hr*ng/mL (41%CV)] resembled levels in human therapeutic trials; whereas canine sapanisertib exposure was reduced when combined with trametinib, a known cytochrome P450 CYP3A4 inducer. Sex differences were not observed for either drug. Side effects upon repeat dosing with either or both drugs may include body weight loss, maldigestion, and cutaneous discoloration. The combination was tolerated without dose limiting toxicity, although clinical laboratory analyses revealed drug-induced acute-phase inflammation, proteinuria, and decreased blood reticulocytes, mild changes not necessitating intervention. Short-term results in dogs with this combination would appear to hold translational promise for clinical trial evaluation to target canine and possibly human melanoma, as well as other cancers having one or both signal transduction pathway activations.

CCL5 production in lung cancer cells leads to an altered immune microenvironment and promotes tumor development.

Current immunotherapies for lung cancer are only effective in a subset of patients. Identifying tumor-derived factors that facilitate immunosuppression offers the opportunity to develop novel strategies to supplement and improve current therapeutics. We sought to determine whether expression of driver oncogenes in lung cancer cells affects cytokine secretion, alters the local immune environment, and influences lung tumor progression. We demonstrate that oncogenic EGFR and KRAS mutations, which are early events in lung tumourigenesis, can drive cytokine and chemokine production by cancer cells. One of the most prominent changes was in CCL5, which was rapidly induced by KRASG12V or EGFRL858R expression, through MAPK activation. Immunocompetent mice implanted with syngeneic KRAS-mutant lung cancer cells deficient in CCL5 have decreased regulatory T cells (Tregs), evidence of T cell exhaustion, and reduced lung tumor burden, indicating tumor-cell CCL5 production contributes to an immune suppressive environment in the lungs. Furthermore, high CCL5 expression correlates with poor prognosis, immunosuppressive regulatory T cells, and alteration to CD8 effector function in lung adenocarcinoma patients. Our data support targeting CCL5 or CCL5 receptors on immune suppressive cells to prevent formation of an immune suppressive tumor microenvironment that promotes lung cancer progression and immunotherapy insensitivity.