Targeted inducible delivery of immunoactivating cytokines reprograms glioblastoma microenvironment and inhibits growth in mouse models.

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor characterized by a strongly immunosuppressive tumor microenvironment (TME) that represents a barrier also for the development of effective immunotherapies. The possibility to revert this hostile TME by immunoactivating cytokines is hampered by the severe toxicity associated with their systemic administration. Here, we exploited a lentiviral vector-based platform to engineer hematopoietic stem cells ex vivo with the aim of releasing, via their tumor-infiltrating monocyte/macrophage progeny, interferon-α (IFN-α) or interleukin-12 (IL-12) at the tumor site with spatial and temporal selectivity. Taking advantage of a syngeneic GBM mouse model, we showed that inducible release of IFN-α within the TME achieved robust tumor inhibition up to eradication and outperformed systemic treatment with the recombinant protein in terms of efficacy, tolerability, and specificity. Single-cell RNA sequencing of the tumor immune infiltrate revealed reprogramming of the immune microenvironment toward a proinflammatory and antitumoral state associated with loss of a macrophage subpopulation shown to be associated with poor prognosis in human GBM. The spatial and temporal control of IL-12 release was critical to overcome an otherwise lethal hematopoietic toxicity while allowing to fully exploit its antitumor activity. Overall, our findings demonstrate a potential therapeutic approach for GBM and set the bases for a recently launched first-in-human clinical trial in patients with GBM.

Interferon gene therapy reprograms the leukemia microenvironment inducing protective immunity to multiple tumor antigens.

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing.

miRNA-126 Orchestrates an Oncogenic Program in B Cell Precursor Acute Lymphoblastic Leukemia.

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.

Angiopoietin 2 expression in the cornea and its control of corneal neovascularisation.

PURPOSE: To define proangiogenic angiopoietin 2 (ANG2) expression and role(s) in human and mouse vascularised corneas. Further, to evaluate the effect of ANG2 inhibition on corneal neovascularisation (CNV). METHODS: CNV was induced in FVB mice by means of intrastromal suture placement. One group of animals was sacrificed 10 days later; corneas were immunostained for ANG2 and compared with (i) mouse non-vascularised corneas and (ii) human vascularised and non-vascularised corneas. A second group of CNV animals was treated systemically with an anti-ANG2 antibody. After 10 days, the corneas were whole-mounted, stained for CD31 and LYVE1 and lymphatic/blood vessels quantified. In another set of experiments, the corneal basal Bowman membrane was either (i) removed or (ii) left in place. After 2 or 10 days the corneas were removed and immunostained for collagen IV, ANG2, CD31, LYVE1, CD11b and MRC1 markers. RESULTS: In human beings and mice, ANG2 is expressed only in the epithelium, and, mildly, in the endothelium, of the avascular cornea. Instead, it is expressed in the epithelium, endothelium and stroma of vascularised corneas. Disruption of the Bowman membrane is associated with a significant increase of (i) ANG2 stromal expression and (ii) proangiogenic macrophage infiltration in the corneal stroma. Finally, blocking ANG2 significantly reduced hemangiogenesis, lymphangiogenesis and macrophage infiltration. CONCLUSIONS: Balancing proper healing and good vision is crucial in the cornea, constantly exposed to potential injuries. In this paper, we suggest the existence of a mechanism regulating the onset of inflammation (and associated CNV) depending on injury severity.

Genetic engineering of hematopoiesis for targeted IFN-α delivery inhibits breast cancer progression.

The immunosuppressive tumor microenvironment represents a major hurdle to cancer therapy. We developed a gene transfer strategy into hematopoietic stem cells (HSCs) to target transgene expression to tumor-infiltrating monocytes/macrophages. Using a combination of transcriptional and microRNA-mediated control, we achieved selective expression of an interferon-α (IFN-α) transgene in differentiated monocytes of human hematochimeric mice. We show that IFN-α transgene expression does not impair engraftment and long-term multilineage repopulation of NSG (NOD/LtSz-scidIL2Rγ(null)) mice by transplanted human HSCs. By providing a source of human cytokines in the mice, we improved the functional reconstitution of human myeloid, natural killer, and T cell lineages, and achieved enhanced immune-mediated clearance of transplanted human breast tumors when hematopoiesis was engineered for tumor-targeted IFN-α expression. By applying our strategy to mouse breast cancer models, we achieved inhibition of tumor progression and experimental metastases in an autologous setting, likely through enhanced generation of effector T cells and their recruitment to the neoplastic tissues. By forcing IFN-α expression in tumor-infiltrating macrophages, we blunted their innate protumoral activity and reprogrammed the tumor microenvironment toward more effective dendritic cell activation and immune effector cell cytotoxicity. Overall, our studies validate the feasibility, safety, and therapeutic potential of a new cancer gene therapy strategy, and open the way to test this approach as adjuvant therapy in advanced breast cancer patients.

miR-511-3p modulates genetic programs of tumor-associated macrophages.

Expression of the mannose receptor (MRC1/CD206) identifies macrophage subtypes, such as alternatively activated macrophages (AAMs) and M2-polarized tumor-associated macrophages (TAMs), which are endowed with tissue-remodeling, proangiogenic, and protumoral activity. However, the significance of MRC1 expression for TAM's protumoral activity is unclear. Here, we describe and characterize miR-511-3p, an intronic microRNA (miRNA) encoded by both mouse and human MRC1 genes. By using sensitive miRNA reporter vectors, we demonstrate robust expression and bioactivity of miR-511-3p in MRC1(+) AAMs and TAMs. Unexpectedly, enforced expression of miR-511-3p tuned down the protumoral gene signature of MRC1(+) TAMs and inhibited tumor growth. Our findings suggest that transcriptional activation of Mrc1 in TAMs evokes a genetic program orchestrated by miR-511-3p, which limits rather than enhances their protumoral functions. Besides uncovering a role for MRC1 as gatekeeper of TAM's protumoral genetic programs, these observations suggest that endogenous miRNAs may operate to establish thresholds for inflammatory cell activation in tumors.

Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis by impairing angiogenesis and disabling rebounds of proangiogenic myeloid cells.

Tumor-infiltrating myeloid cells convey proangiogenic programs that counteract the efficacy of antiangiogenic therapy. Here, we show that blocking angiopoietin-2 (ANG2), a TIE2 ligand and angiogenic factor expressed by activated endothelial cells (ECs), regresses the tumor vasculature and inhibits progression of late-stage, metastatic MMTV-PyMT mammary carcinomas and RIP1-Tag2 pancreatic insulinomas. ANG2 blockade did not inhibit recruitment of MRC1(+) TIE2-expressing macrophages (TEMs) but impeded their upregulation of Tie2, association with blood vessels, and ability to restore angiogenesis in tumors. Conditional Tie2 gene knockdown in TEMs was sufficient to decrease tumor angiogenesis. Our findings support a model wherein the ANG2-TIE2 axis mediates cell-to-cell interactions between TEMs and ECs that are important for tumor angiogenesis and can be targeted to induce effective antitumor responses.

Gene expression profiles identify inflammatory signatures in dendritic cells.

Dendritic cells (DCs) constitute a heterogeneous group of antigen-presenting leukocytes important in activation of both innate and adaptive immunity. We studied the gene expression patterns of DCs incubated with reagents inducing their activation or inhibition. Total RNA was isolated from DCs and gene expression profiling was performed with oligonucleotide microarrays. Using a supervised learning algorithm based on Random Forest, we generated a molecular signature of inflammation from a training set of 77 samples. We then validated this molecular signature in a testing set of 38 samples. Supervised analysis identified a set of 44 genes that distinguished very accurately between inflammatory and non inflammatory samples. The diagnostic performance of the signature genes was assessed against an independent set of samples, by qRT-PCR. Our findings suggest that the gene expression signature of DCs can provide a molecular classification for use in the selection of anti-inflammatory or adjuvant molecules with specific effects on DC activity.

Novel members of the family Micromonosporaceae, Rugosimonospora acidiphila gen. nov., sp. nov. and Rugosimonospora africana sp. nov.

Two novel Gram-positive-staining, acidophilic strains were isolated from soil samples. Both show typical features of filamentous actinomycetes. On the basis of 16S rRNA gene sequence analysis, the strains are members of the family Micromonosporaceae. The two strains contain hydroxydiaminopimelic acid, glycine, alanine and glutamic acid in the peptidoglycan. Fatty acid profiles clearly differentiate the two strains: cyclohexyl C(17 : 0), i-C(16 : 0) and ai-C(17 : 0) are predominant in Delta1(T), while the major components for Delta3(T) are ai-C(17 : 0) and i-C(16 : 0). The two strains also differ in their major menaquinones, MK-9(H(8), H(4), H(6)) for Delta1(T) and MK-9(H(8), H(6)) for Delta3(T), and in phospholipid patterns; Delta1(T) displays phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, methyl phosphatidylethanolamine and an unknown aminophospholipid, while Delta3(T) also contains minor amounts of several unknown phospholipids in addition to these phospholipids. The whole-cell sugars of both strains are galactose, arabinose and xylose. The G+C content of the DNA is 72.7 mol% for Delta1(T) and 71.9 mol% for Delta3(T). On the basis of chemotaxonomic, physiological and phylogenetic data, we propose Rugosimonospora gen. nov. to accommodate the two strains, with the description of Rugosimonospora acidiphila gen. nov., sp. nov. (the type species; type strain Delta1(T) =DSM 45227(T) =NBRC 104874(T)) and Rugosimonospora africana sp. nov. (type strain Delta3(T) =DSM 45228(T) =NBRC 104875(T)).