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Ewing sarcoma (ES) is the second most common pediatric bone cancer. Despite recent advances in the treatment, patients with metastatic tumors have dismal prognosis and hence novel therapies are urgently needed to combat this cancer. A recent study has shown that phosphoinositide-3 kinase (PI3K) inhibitors can synergistically increase sensitivity to bromodomain and extraterminal domain inhibitors in ES cells and therefore combined inhibition of PI3K and bromodomain and extraterminal domain bromodomain proteins might provide benefit in this cancer. Herein, we have investigated the efficacy of dual PI3K/BRD4 inhibitors, SF2523 and SF1126, for their antitumor activity in ES cell lines. The effect of SF1126 and SF2523 on cell viability and PI3K signaling was assessed on a panel of human ES cell lines. To evaluate the antitumor activity of SF1126, A673 cells were injected intrafemorally into RAG-2-/- mice and treated with 50 mg/kg SF1126 6 days per week, for 30 days. Both SF1126 and SF2523 decreased cell survival and inhibited phosphorylation of AKT in human ES cell lines. In vivo, SF1126 showed a significant reduction in tumor volume. These results suggest that dual PI3K/BRD4 inhibitor, SF1126, has antitumor activity in ES models.

Development of small molecule compounds that target several cancer drivers has shown great therapeutic potential. Here, we developed a new generation of highly potent thienopyranone (TP)-based inhibitors for the BET bromodomains (BDs) of the transcriptional regulator BRD4 that have the ability to simultaneously bind to phosphatidylinositol-3 kinase (PI3K) and/or cyclin-dependent kinases 4/6 (CDK4/6). Analysis of the crystal structures of the complexes, NMR titration experiments and IC50 measurements reveal the molecular basis underlying the inhibitory effects and selectivity of these compounds toward BDs of BRD4. The inhibitors show robust cytotoxic effects in multiple cancer cell lines and induce cell-cycle arrest and apoptosis. We further demonstrate that concurrent disruption of the acetyllysine binding function of BRD4 and the kinase activities of PI3K and CDK4/6 by the TP inhibitor improves efficacy in several cancer models. Together, these findings provide further compelling evidence that these multi-action inhibitors are efficacious and more potent than single inhibitory chemotypes.

Macrophages (MΦ) play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that an MΦ Syk-PI3K axis drives polarization of immunosuppressive MΦs that establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacologic inhibition of Syk and/or PI3Kγ in MΦs promotes a proinflammatory MΦ phenotype, restores CD8+ T-cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for transposase-accessible 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-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs 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.

Macrophages (MΘs) 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 IL4-driven MΘ transcriptional programming and MΘ-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 MΘ genes, which promote immunosuppression in TME. Pharmacologic 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 MΘs; restores CD8+ 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 MΘ-dependent immunosuppressive TME.

Dysregulation of the seven-transmembrane (7TM) receptor Smoothened (SMO) and other components of the Hedgehog (Hh) signaling pathway contributes to the development of cancers including basal cell carcinoma (BCC) and medulloblastoma (MB). However, SMO-specific antagonists produced mixed results in clinical trials, marked by limited efficacy and high rate of acquired resistance in tumors. Here we discovered that Nilotinib, an approved inhibitor of several kinases, possesses an anti-Hh activity, at clinically achievable concentrations, due to direct binding to SMO and inhibition of SMO signaling. Nilotinib was more efficacious than the SMO-specific antagonist Vismodegib in inhibiting growth of two Hh-dependent MB cell lines. It also reduced tumor growth in subcutaneous MB mouse xenograft model. These results indicate that in addition to its known activity against several tyrosine-kinase-mediated proliferative pathways, Nilotinib is a direct inhibitor of the Hh pathway. The newly discovered extension of Nilotinib's target profile holds promise for the treatment of Hh-dependent cancers.

In this study, we investigated the effects of the dual phosphatidylinositol 3-kinase/mechanistic target of rapamycin (PI3K/MTOR) inhibitor dactolisib (NVP-BEZ235), the PI3K/MTOR/bromodomain-containing protein 4 (BRD4) inhibitor SF2523, and the bromodomain and extra terminal domain inhibitor JQ1 on the productive infection of primary macrophages with human immunodeficiency type-1 (HIV). These inhibitors did not alter the initial susceptibility of macrophages to HIV infection. However, dactolisib, JQ1, and SF2523 all decreased HIV replication in macrophages in a dose-dependent manner via degradation of intracellular HIV through autophagy. Macrophages treated with dactolisib, JQ1, or SF2523 displayed an increase in LC3B lipidation combined with SQSTM1 degradation without inducing increased cell death. LC3B-II levels were further increased in the presence of pepstatin A suggesting that these inhibitors induce autophagic flux. RNA interference for ATG5 and ATG7 and pharmacological inhibitors of autophagosome-lysosome fusion and of lysosomal hydrolases all blocked the inhibition of HIV. Thus, we demonstrate that the mechanism of PI3K/MTOR and PI3K/MTOR/BRD4 inhibitor suppression of HIV requires the formation of autophagosomes, as well as their subsequent maturation into autolysosomes. These data provide further evidence in support of a role for autophagy in the control of HIV infection and open new avenues for the use of this class of drugs in HIV therapy.

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.

Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. We investigated synovial, vascular, stromal, and cartilage changes in response to a single induced hemarthrosis in the FVIII-deficient mouse. We found soft-tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture. While soft-tissue changes were rapidly reversible, abnormal vascularity persisted for months and, surprisingly, was also seen in uninjured joints. Vascular changes in FVIII-deficient mice involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105), and vascular endothelial growth factor, as well as alterations of joint perfusion as determined by in vivo imaging. Vascular architecture changes and pronounced expression of α-SMA appeared unique to hemophilia, as these were not found in joint tissue obtained from mouse models of rheumatoid arthritis and osteoarthritis and from patients with the same conditions. Evidence that vascular changes in hemophilia were significantly associated with bleeding and joint deterioration was obtained prospectively by dynamic in vivo imaging with musculoskeletal ultrasound and power Doppler of 156 joints (elbows, knees, and ankles) in a cohort of 26 patients with hemophilia at baseline and during painful episodes. These observations support the hypothesis that vascular remodeling contributes significantly to bleed propagation and development of hemophilic arthropathy. Based on these findings, the development of molecular targets for angiogenesis inhibition may be considered in this disease.

Knockout serum replacement (KOSR) is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML) cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors--imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.

BackgroundFcγ receptors mediate important biological signals in myeloid cells including the ingestion of microorganisms through a process of phagocytosis. It is well-known that Fcγ receptor (FcγR) crosslinking induces the tyrosine phosphorylation of CBL which is associated with FcγR mediated phagocytosis, however how signaling molecules coordinate to desensitize these receptors is unclear. An investigation of the mechanisms involved in receptor desensitization will provide new insight into potential mechanisms by which signaling molecules may downregulate tyrosine phosphorylation dependent signaling events to terminate important signaling processes.ResultsUsing the U937IF cell line, we observed that FcγR1 crosslinking induces the tyrosine phosphorylation of CBL, which is maximal at 5 min. followed by a kinetic pattern of dephosphorylation. An investigation of the mechanisms involved in receptor desensitization revealed that pretreatment of U937IF or J774 cells with PMA followed by Fcγ receptor crosslinking results in the reduced tyrosine phosphorylation of CBL and the abrogation of downstream signals, such as CBL-CRKL binding, Rac-GTP activation and the phagocytic response. Pretreatment of J774 cells with GF109203X, a PKC inhibitor was observed to block dephosphorylation of CBL and rescued the phagocytic response. We demonstrate that the PKC induced desensitization of FcγR/ phagocytosis is associated with the inactivation of Rac-GTP, which is deactivated in a hematopoietic specific phosphatase SHP1 dependent manner following ITAM stimulation. The effect of PKC on FcγR signaling is augmented by the transfection of catalytically active SHP1 and not by the transfection of catalytic dead SHP1 (C124S).ConclusionsOur results suggest a functional model by which PKC interacts with SHP1 to affect the phosphorylation state of CBL, the activation state of Rac and the negative regulation of ITAM signaling i.e. Fcγ receptor mediated phagocytosis. These findings suggest a mechanism for Fcγ receptor desensitization by which a serine-threonine kinase e.g. PKC downregulates tyrosine phosphorylation dependent signaling events via the reduced tyrosine phosphorylation of the complex adapter protein, CBL.

Brain tumors are the most common solid tumor diagnosed in childhood that account for significant morbidity and mortality. New therapies are urgently needed; hence, we conducted the first ever prospective open-label phase II trials of the biological response modifier, poly-ICLC, in children with brain tumors. Poly-ICLC is a synthetic double-stranded RNA that has direct antiviral, antineoplastic, and immune adjuvant effects. A total of 47 children representing a variety of brain tumor histopathologic subtypes were treated with poly-ICLC. On the basis of the results of the initial phase II trial, an expanded prospective phase II trial in low-grade glioma (LGG) has been initiated. MRI was used to acquire volume-based measures of tumor response. No dose-limiting toxicities have been observed. In the initial study 3 of 12 subjects with progressive high-grade gliomas (HGGs) responded, and 2 of 4 children with progressive LGG experienced stable disease for 18 to 24 months. In the follow-up LGG phase II study, 2 of 5 LGG patients were stable over 18 months, with 1 stable for 6 months. Overall 5 of 10 LGG patients have responded. On the basis of low toxicity and the promising LGG response, poly-ICLC may be effective for childhood LGG, and the results justify biomarker studies for personalization of poly-ICLC as a single agent or adjuvant.

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.