Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4β1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4β1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis.

Tumor growth, progression, and response to the hypoxic tumor microenvironment involve the action of hypoxia-inducible transcription factors, HIF1 and HIF2. HIF is a heterodimeric transcription factor containing an inducible HIFa subunit and a constitutively expressed HIFb subunit. The signaling pathways operational in macrophages regulating hypoxia-induced HIFa stabilization remain the subject of intense investigation. Here, it was discovered that the PTEN/PI3K/AKT signaling axis controls hypoxia-induced HIF1a (HIF1A) and HIF2a (EPAS1) stability in macrophages. Using genetic mouse models and pan-PI3K as well as isoform-specific inhibitors, inhibition of the PI3K/AKT pathway blocked the accumulation of HIFa protein and its primary transcriptional target VEGF in response to hypoxia. Moreover, blocking the PI3K/AKT signaling axis promoted the hypoxic degradation of HIFa via the 26S proteasome. Mechanistically, a macrophage-dominant PI3K isoform (p110g) directed tumor growth, angiogenesis, metastasis, and the HIFa/VEGF axis. Moreover, a pan-PI3K inhibitor (SF1126) blocked tumor-induced angiogenesis and inhibited VEGF and other proangiogenic factors secreted by macrophages. These data define a novel molecular mechanism by which PTEN/PI3K/AKT regulates the proteasome-dependent stability of HIFa under hypoxic conditions, a signaling pathway in macrophages that controls tumor-induced angiogenesis and metastasis.

Macrophages (MÂs) are key immune infiltrates in solid tumors and serve as major drivers behind tumor growth, immune suppression, and inhibition of adaptive immune responses in the tumor microenvironment (TME). Bromodomain and extraterminal (BET) protein, BRD4, which binds to acetylated lysine on histone tails, has recently been reported to promote gene transcription of proinflammatory cytokines but has rarely been explored for its role in IL4driven MÂ transcriptional programming and MÂ-mediated immunosuppression in the TME. Herein, we report that BET bromodomain inhibitor, JQ1, blocks association of BRD4 with promoters of arginase and other IL4-driven MÂ genes, which promote immunosuppression in TME. Pharmacolog-ic inhibition of BRD4 using JQ1 and/or PI3K using dual PI3K/BRD4 inhibitor SF2523 (previously reported by our group as a potent inhibitor to block tumor growth and metastasis in various cancer models) suppresses tumor growth in syngeneic and spontaneous murine cancer models; reduces infiltration of myeloid-derived suppressor cells; blocks polarization of immunosuppressive MÂs; restores CD8 þ T-cell activity; and stimulates antitumor immune responses. Finally, our results suggest that BRD4 regulates the immunosuppressive myeloid TME, and BET inhibitors and dual PI3K/BRD4 inhibitors are therapeutic strategies for cancers driven by the MÂ-dependent immunosuppressive TME. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis):

◥ Macrophages (MÈ) play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MÈs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kg has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that an MÈ Syk-PI3K axis drives polarization of immunosuppressive MÈs that establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacologic inhibition of Syk and/or PI3Kg in MÈs promotes a proinflammatory MÈ phenotype, restores CD8 þ T-cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for transposaseaccessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow-derived macrophages (BMDM) show that inhibition of Syk kinase promotes activation and binding of NF-kB motif in Syk MC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MÈs to suppress tumor growth. Finally, we have developed in silico the "first-in-class" dual Syk/ PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.

Sonic hedgehog (SHH) medulloblastoma (MB) subtype is driven by a proliferative CD15+ tumor propagating cell (TPC), also considered in the literature as a putative cancer stem cell (CSC). Despite considerable research, much of the biology of this TPC remains unknown. We report evidence that phosphatase and tensin homolog (PTEN) and phosphoinositide 3-kinase (PI-3K) play a crucial role in the propagation, survival and potential response to therapy in this CD15+ CSC/TPC-driven malignant disease. Using the ND2-SmoA1 transgenic mouse model for MB, mouse genetics and patient-derived xenografts (PDXs), we demonstrate that the CD15+TPCs are 1) obligately required for SmoA1Tg-driven tumorigenicity 2) regulated by PTEN and PI-3K signaling 3) selectively sensitive to the cytotoxic effects of pan PI-3K inhibitors in vitro and in vivo but resistant to chemotherapy 4) in the SmoA1Tg mouse model are genomically similar to the SHH human MB subgroup. The results provide the first evidence that PTEN plays a role in MB TPC signaling and biology and that PI-3K inhibitors target and suppress the survival and proliferation of cells within the mouse and human CD15+ cancer stem cell compartment. In contrast, CD15+ TPCs are resistant to cisplatinum, temozolomide and the SHH inhibitor, NVP-LDE-225, agents currently used in treatment of medulloblastoma. These studies validate the therapeutic efficacy of pan PI-3K inhibitors in the treatment of CD15+ TPC dependent medulloblastoma and suggest a sequential combination of PI-3K inhibitors and chemotherapy will have augmented efficacy in the treatment of this disease.