The FLRT3-UNC5B checkpoint pathway inhibits T cell-based cancer immunotherapies.

Cancers exploit coinhibitory receptors on T cells to escape tumor immunity, and targeting such mechanisms has shown remarkable clinical benefit, but in a limited subset of patients. We hypothesized that cancer cells mimic noncanonical mechanisms of early development such as axon guidance pathways to evade T cell immunity. Using gain-of-function genetic screens, we profiled axon guidance proteins on human T cells and their cognate ligands and identified fibronectin leucine-rich transmembrane protein 3 (FLRT3) as a ligand that inhibits T cell activity. We demonstrated that FLRT3 inhibits T cells through UNC5B, an axon guidance receptor that is up-regulated on activated human T cells. FLRT3 expressed in human cancers favored tumor growth and inhibited CAR-T and BiTE + T cell killing and infiltration in humanized cancer models. An FLRT3 monoclonal antibody that blocked FLRT3-UNC5B interactions reversed these effects in an immune-dependent manner. This study supports the concept that axon guidance proteins mimic T cell checkpoints and can be targeted for cancer immunotherapy.

TPI-287, a new taxane family member, reduces the brain metastatic colonization of breast cancer cells.

Brain metastases of breast and other cancers remain resistant to chemotherapeutic regimens that are effective systemically, in part due to the blood-brain barrier. We report that TPI-287, a new microtubule-stabilizing agent, displays in vitro cytotoxic activity similar to taxanes and epothilones. Unlike the taxanes, TPI-287 is permeable through the blood-brain barrier. Brain-to-plasma ratios of TPI-287 after a single injection typically exceeded one and were as high as 63.8 in the rat and 14.1 in the mouse. A brain-tropic derivative of the MDA-MB-231 triple-negative breast cancer cell line, 231-BR, was used to test whether TPI-287 may be efficacious at preventing or treating brain metastases. TPI-287 had growth inhibitory effects comparable with paclitaxel when 231-BR tumor cells were injected into the mammary fat pad. Brain metastatic colonization was determined by intracardiac injection of 231-BR cells, with treatment beginning on day 3 to 4 postinjection, culminating in a histologic count of brain metastases in brains necropsied days 25 to 28 postinjection. In this assay, paclitaxel, ixabepilone, and nab paclitaxel did not have significant inhibitory activity. TPI-287 was ineffective in the same assay using a 6 mg/kg every week schedule; however an 18 mg/kg dose delivered on days 3, 7, and 11 significantly reduced the outgrowth of brain metastases (55% reduction, P = 0.028) and reduced proliferation in brain metastases (16% reduction, P = 0.008). When TPI-287 treatment was delayed until days 18, 22, and 26 postinjection, efficacy was reduced (17% reduction, not significant). These data suggest that TPI-287 may have efficacy when administered early in the course of the disease.

Opposing effects of pigment epithelium-derived factor on breast cancer cell versus neuronal survival: implication for brain metastasis and metastasis-induced brain damage.

Brain metastases are a significant cause of morbidity and mortality for patients with cancer, yet preventative and therapeutic options remain an unmet need. The cytokine pigment epithelium-derived factor (PEDF) is downregulated in resected human brain metastases of breast cancer compared with primary breast tumors, suggesting that restoring its expression might limit metastatic spread. Here, we show that outgrowth of large experimental brain metastases from human 231-BR or murine 4T1-BR breast cancer cells was suppressed by PEDF expression, as supported by in vitro analyses as well as direct intracranial implantation. Notably, the suppressive effects of PEDF were not only rapid but independent of the effects of this factor on angiogenesis. Paralleling its cytotoxic effects on breast cancer cells, PEDF also exerted a prosurvival effect on neurons that shielded the brain from tumor-induced damage, as indicated by a relative 3.5-fold reduction in the number of dying neurons adjacent to tumors expressing PEDF. Our findings establish PEDF as both a metastatic suppressor and a neuroprotectant in the brain, highlighting its role as a double agent in limiting brain metastasis and its local consequences.

Reactive glia are recruited by highly proliferative brain metastases of breast cancer and promote tumor cell colonization.

Interactions between tumor cells and the microenvironment are crucial to tumor formation and metastasis. The central nervous system serves as a "sanctuary" site for metastasis, resulting in poor prognosis in diagnosed patients. The incidence of brain metastasis is increasing; however, little is known about interactions between the brain and metastatic cells. Brain pathology was examined in an experimental model system of brain metastasis, using a subline of MDA-MB-231 human breast cancer cells. The results were compared with an analysis of sixteen resected human brain metastases of breast cancer. Experimental metastases formed preferentially in specific brain regions, with a distribution similar to clinical cases. In both the 231-BR model, and in human specimens, Ki67 expression indicated that metastases were highly proliferative (approximately 50%). Little apoptosis was observed in either set of tumors. In the model system, metastases elicited a brain inflammatory response, with extensive reactive gliosis surrounding metastases. Similarly, large numbers of glial cells were found within the inner tumor mass of human brain metastases. In vitro co-cultures demonstrated that glia induced a approximately 5-fold increase in metastatic cell proliferation (P<0.001), suggesting that brain tissue secretes factors conducive to tumor cell growth. Molecules used to signal between tumor cells and the surrounding glia could provide a new avenue of therapeutic targets for brain metastases.

Neogenin is expressed on neurogenic and gliogenic progenitors in the embryonic and adult central nervous system.

The Netrin/RGMa receptor, Neogenin, has recently been identified on neuronal and gliogenic progenitors, including radial glia in the embryonic mouse cortex and ganglionic eminences, respectively [Fitzgerald, D.P., Cole, S.J., Hammond, A., Seaman, C., Cooper, H.M., 2006a. Characterization of Neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain. Neuroscience 142, 703-716]. Here we have undertaken a detailed analysis of Neogenin expression in the embryonic mouse central nervous system at key developmental time points. We demonstrate that Neogenin protein is present on actively dividing neurogenic precursors during peak phases of neurogenesis (embryonic days 12.5-14.5) in the forebrain, midbrain and hindbrain. Furthermore, we show that Neogenin protein is localized to the cell bodies and glial processes of neurogenic radial glial populations in all these regions. We have also observed Neogenin on gliogenic precursors within the subventricular zones of the forebrain late in development (embryonic day 17.5). Adult neural stem cells found in the subventricular zone of the lateral ventricle of the rodent forebrain are direct descendants of the embryonic striatal radial glial population. Here we show that Neogenin expression is maintained in the neural stem cell population of the adult mouse forebrain. In summary, this study demonstrates that Neogenin expression is a hallmark of many neural precursor populations (neurogenic and gliogenic) in both the embryonic and adult mammalian central nervous system.

Localization of Neogenin protein during morphogenesis in the mouse embryo.

Neogenin, a close relative of the axon guidance receptor DCC, has been shown to be a receptor for members of the Netrin and Repulsive Guidance Molecule families. Recent studies have begun to uncover a role for Neogenin in organogenesis. Here we examine the localization of Neogenin protein in the developing mouse embryo (embryonic day 14.5) when organogenesis is progressing rapidly. We observe that Neogenin protein is restricted to distinct tissue layers within a given organ. In some embryonic epithelia such as the gut and pancreas, Neogenin protein is predominantly polarized to the basal surfaces of the epithelial cells. In contrast, Neogenin is restricted to mesenchymal cells within the lung and kidney. Neogenin is also seen in differentiating skeletal muscle and condensing cartilage throughout the embryo, and in the trigeminal and dorsal root ganglia of the peripheral nervous system. This study supports the emerging role for Neogenin as a key receptor in the establishment of the morphological architecture in many developing organ systems.

Transcription activates repressed domains in the Drosophila bithorax complex.

A series of mutations have been recovered in the bithorax complex of D. melanogaster that transform the first segment of the abdomen into a copy of the second or third abdominal segment. These dominant Ultraabdominal alleles are all associated with P element insertions which are transcribed in the first abdominal segment. The transcripts proceed past the end of the P element for up to 50 kb, extending through the regulatory regions for the second and third abdominal segments. Blocking transcription from the P element promoter reverts the mutant phenotype. Previously identified Ultraabdominal alleles, not associated with P elements, also show abnormal transcription of the same region.