Dual-activity PI3K-BRD4 inhibitor for the orthogonal inhibition of MYC to block tumor growth and metastasis.

MYC is a major cancer driver but is documented to be a difficult therapeutic target itself. Here, we report on the biological activity, the structural basis, and therapeutic effects of the family of multitargeted compounds that simultaneously disrupt functions of two critical MYC-mediating factors through inhibiting the acetyllysine binding of BRD4 and the kinase activity of PI3K. We show that the dual-action inhibitor impairs PI3K/BRD4 signaling in vitro and in vivo and affords maximal MYC down-regulation. The concomitant inhibition of PI3K and BRD4 blocks MYC expression and activation, promotes MYC degradation, and markedly inhibits cancer cell growth and metastasis. Collectively, our findings suggest that the dual-activity inhibitor represents a highly promising lead compound for the development of novel anticancer therapeutics.

MDM2 regulates hypoxic hypoxia-inducible factor 1α stability in an E3 ligase, proteasome, and PTEN-phosphatidylinositol 3-kinase-AKT-dependent manner.

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1ß subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α-HIF1ß heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics.

PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment.

Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN(fl/fl) mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo.

Anti-tumor effect of a novel PI3-kinase inhibitor, SF1126, in (12) V-Ha-Ras transgenic mouse glioma model.

BACKGROUND: Growth factor mediated activation of RAS-MAP-kinase and PI3-kinase-AKT pathways are critical for the pathogenesis of glioblastoma. The attenuation of PI3-kinase/AKT signaling will be effective in regulating the tumorigenic phenotypes of the glioma cells. METHODS: Glioma cells derived from the brain of the (12) V-Ha-Ras transgenic mice were used to study the effect of PI-3 kinase inhibitor SF1126 on activation of AKT and ERK signaling, proliferation, vitronectin mediated migration and changes in the distribution of cortical actin on vitronectin in the glioma cells in vitro. The anti-tumor effects of SF1126 were also tested in vivo using pre-established tumors (subcutaneous injection of the glioma cells from (12) V-Ha-Ras transgenic mice) in a mouse xenograft model. RESULTS: Our results demonstrate that treatment of LacZ+, GFAP + and PCNA + (12) V-Ras Tg transformed astrocytes with SF1126 and LY294002 blocked the activation of AKT as well as EGF-induced phospho-ERK. Most notably, treatment of SF1126 blocked integrin-dependent migration in transwell and scratch assays and caused a significant change in the organization and distribution of cortical actin on vitronectin in the glioma cells. Moreover, SF1126 treatment inhibited in vitro proliferation of these cells and in vivo growth of pre-established subcutaneous tumors in a xenograft model. CONCLUSION: The present study validate the potent anti-proliferative and anti-migratory activity of SF1126, in a V(12) Ras oncogene driven glioma model and suggest that this effect is mediated potentially through a combined attenuation of PI3-kinase and MAP-kinase signaling pathways.

PTEN status mediates 2ME2 anti-tumor efficacy in preclinical glioblastoma models: role of HIF1α suppression.

Glioblastoma (GBM) is the most common brain cancer and is highly lethal in both adults and children. 2-methoxyestradiol (2ME2) is a microtubule inhibitor that potently inhibits HIF1α, GBM angiogenesis and tumor growth in preclinical models. In patients, 2ME2 exhibits low toxicity and promising but inconsistent efficacy. Given its preclinical potency and its tolerability in patients, we sought to determine whether 2ME2 therapy could be enhanced by addressing resistance via combination therapy, and with biomarkers to identify responsive glioma subgroups. We demonstrate that the PTEN-PI3K axis regulates HIF1α in glioma models. We utilized isogenic-pairs of glioma cell lines, deficient in PTEN or stably reconstituted with PTEN, to determine the role of PTEN in 2ME2 sensitivity in vitro and in vivo. Chou-Talalay synergy studies reveal significant synergy when a pan-PI3K inhibitor is combined with 2ME2. This synergistic activity was correlated with a synergistic suppression of HIF1α accumulation under hypoxic conditions in glioma models. In vivo, 2ME2 markedly inhibited tumor-induced angiogenesis and significantly reduced tumor growth only in a PTEN reconstituted GBM models in both subcutaneous and orthotopic intracranial mouse models. Collectively, these results: (1) suggest that PTEN status predicts sensitivity to 2ME2 and (2) justify exploration of 2ME2 combined with pan-PI3K inhibitors for the treatment of this intractable brain cancer.

A Novel Affinity Engineered Anti-CD47 Antibody With Improved Therapeutic Index That Preserves Erythrocytes and Normal Immune Cells.

Therapeutic blockade of the CD47/SIRPα axis by small molecules or monoclonal antibodies (mAbs) is a proven strategy to enhance macrophages-mediated anti-tumor activity. However, this strategy has been hampered by elevated on-target toxicities and rapid clearance due to the extensive CD47 expression on normal cells ("antigen sink") such as red blood cells (RBCs). To address these hurdles, we report on the development of STI-6643, an affinity-engineered fully human anti-CD47 IgG4 antibody with negligible binding to normal cells. STI-6643 exhibited no hemagglutination activity on human RBCs at concentrations up to 300 µg/mL yet specifically blocked the CD47/SIPRα interaction. Of particular interest, STI-6643 preserved T cell functionality in vitro and showed significantly lower immune cell depletion in vivo in contrast to three previously published competitor reference anti-CD47 clones Hu5F9, AO-176 and 13H3. In cynomolgus monkeys, STI-6643 was well-tolerated at the highest dose tested (300 mg/kg/week) and provided favorable clinical safety margins. Finally, STI-6643 displayed comparable anti-tumor activity to the high-affinity reference clone Hu5F9 in a RAJI-Fluc xenograft tumor model as monotherapy or in combination with anti-CD20 (rituximab) or anti-CD38 (daratumumab) mAbs. These data suggest that STI-6643 possesses the characteristics of an effective therapeutic candidate given its potent anti-tumor activity and low toxicity profile.

Macrophage Syk-PI3Kγ Inhibits Antitumor Immunity: SRX3207, a Novel Dual Syk-PI3K Inhibitory Chemotype Relieves Tumor Immunosuppression.

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.

SF2523: Dual PI3K/BRD4 Inhibitor Blocks Tumor Immunosuppression and Promotes Adaptive Immune Responses in Cancer.

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.

Rac2 is required for alternative macrophage activation and bleomycin induced pulmonary fibrosis; a macrophage autonomous phenotype.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by cellular phenotype alterations and deposition of extracellular matrix proteins. The alternative activation of macrophages in the lungs has been associated as a major factor promoting pulmonary fibrosis, however the mechanisms underlying this phenomenon are poorly understood. In the present study, we have defined a molecular mechanism by which signals transmitted from the extracellular matrix via the α4ß1 integrin lead to the activation of Rac2 which regulates alternative macrophage differentiation, a signaling axis within the pulmonary macrophage compartment required for bleomycin induced pulmonary fibrosis. Mice deficient in Rac2 were protected against bleomycin-induced fibrosis and displayed diminished collagen deposition in association with lower expression of alternatively activated profibrotic macrophage markers. We have demonstrated a macrophage autonomous process by which the injection of M2 and not M1 macrophages restored the bleomycin induced pulmonary fibrosis susceptibility in Rac2-/- mice, establishing a critical role for a macrophage Rac2 signaling axis in the regulation of macrophage differentiation and lung fibrosis in vivo. We also demonstrate that markers of alternative macrophage activation are increased in patients with IPF. Taken together, these studies define an important role for an integrin-driven Rac2 signaling axis in macrophages, and reveal that Rac2 activation is required for polarization of macrophages towards a profibrotic phenotype and progression of pulmonary fibrosis in vivo.

Association of high microvessel αvß3 and low PTEN with poor outcome in stage 3 neuroblastoma: rationale for using first in class dual PI3K/BRD4 inhibitor, SF1126.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvß3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvß3 was expressed on 68% of microvessels in MYCN-amplified stage 3 neuroblastomas, but only on 34% (means) in MYCN-non-amplified tumors (p < 0.001; n = 54). PTEN, a tumor suppressor involved in αvß3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvß3 was expressed on 60% of tumor microvessels when PTEN was negative or focal, as compared to 32% of microvessels in tumors with diffuse PTEN expression (p < 0.001). In a MYCN transgenic mouse model, loss of one allele of PTEN promoted tumor growth, illustrating the potential role of PTEN in neuroblastoma pathogenesis. Interestingly, we report the novel dual PI-3K/BRD4 activity of SF1126 (originally developed as an RGD-conjugated pan PI3K inhibitor). SF1126 inhibits BRD4 bromodomain binding to acetylated lysine residues with histone H3 as well as PI3K activity in the MYCN amplified neuroblastoma cell line IMR-32. Moreover, SF1126 suppressed MYCN expression and MYCN associated transcriptional activity in IMR-32 and CHLA136, resulting in overall decrease in neuroblastoma cell viability. Finally, treatment of neuroblastoma tumors with SF1126 inhibited neuroblastoma growth in vivo. These data suggest integrin αvß3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvß3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma.

Single Agent and Synergistic Activity of the "First-in-Class" Dual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma.

Deregulated PI3K/AKT/mTOR, Ras/Raf/MAPK, and c-Myc signaling pathways are of prognostic significance in hepatocellular carcinoma (HCC). Sorafenib, the only drug clinically approved for patients with advanced HCC, blocks the Ras/Raf/MAPK pathway but it does not inhibit the PI3K/AKT/mTOR pathway or c-Myc activation. Hence, there is an unmet medical need to identify potent PI3K/BRD4 inhibitors, which can be used either alone or in combination with sorafenib to treat patients with advanced HCC. Herein, we show that SF1126 (pan PI3K/BRD4 inhibitor) as single agent or in combination with sorafenib inhibited proliferation, cell cycle, apoptosis, and multiple key enzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK pathway in Hep3B, HepG2, SK-Hep1, and Huh7 HCC cell lines. We demonstrate that the active moiety of the SF1126 prodrug LY294002 binds to and blocks BRD4 interaction with the acetylated histone-H4 chromatin mark protein and displaced BRD4 coactivator protein from the transcriptional start site of MYC in Huh7 and SK-Hep1 HCC cell lines. Moreover, SF1126 blocked expression levels of c-Myc in HCC cells. Treatment of SF1126 either alone or in combination with sorafenib showed significant antitumor activity in vivo Our results establish that SF1126 is a dual PI3K/BRD4 inhibitor. This agent has completed a phase I clinical trial in humans with good safety profile. Our data support the potential future consideration of a phase II clinical trial of SF1126, a clinically relevant dual "first-in-class" PI3K/BRD4 inhibitor in advanced HCC, and a potential combination with sorafenib. Mol Cancer Ther; 15(11); 2553-62. ©2016 AACR.

PI-3K Inhibitors Preferentially Target CD15+ Cancer Stem Cell Population in SHH Driven Medulloblastoma.

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.

Pan-PI-3 kinase inhibitor SF1126 shows antitumor and antiangiogenic activity in renal cell carcinoma.

PURPOSE: SF1126 is a vascular-targeted pan-PI-3K inhibitor prodrug with antitumor and antiangiogenic activity and has completed phase I clinical trial in solid tumors and B-cell malignancies. In this study, we investigated the effect of SF1126 on hypoxic HIF-1α/HIF-2α stability as well as on antitumor and/or antiangiogenic activity in renal cell carcinoma (RCC) models in vitro and in vivo. METHODS: The effect of SF1126 on hypoxic HIF-1α/HIF-2α protein stability, antitumor and antiangiogenic activity was studied on VHL-null (786-0) and VHL-WT (Caki) RCC cells. RESULTS: Our data demonstrate that SF1126 treatment abrogates the stabilization of HIF-2α in 786-0 (VHL-mutated) RCC cell line under normoxic and hypoxic conditions. Similarly, hypoxic stabilization of HIF-1α and its activity were also suppressed following SF1126 treatment in Caki cell line (VHL-WT). Herein, we provide mechanistic evidence that HIF-2α can be degraded in cytoplasm under hypoxic conditions via the 26S proteasome and that MDM2 is the E3 ligase which induces the hypoxic degradation of HIF-2α in PI-3K-dependent manner in VHL-deficient RCC cells. Moreover, SF1126 administered to RCC-xenografted mice at 25 mg/kg/dose subcutaneously three times per week for 3 weeks results in marked inhibition of tumor growth (>90 % inhibition) (P < 0.05). Consistent with SF1126 treatment's effects on HIF-1α/HIF-2α, microvessel density analysis of Caki and 786-0 tumor tissues demonstrated that SF1126 has potent antiangiogenic activity in vivo. Finally, SF1126 caused a profound inhibition of integrin-mediated migration and blocked the integrin-induced conversion of GDP-Rac1 to its GTP-bound active state. CONCLUSIONS: These results validate the in vivo efficacy of SF1126 as a clinically viable antiangiogenic, pan-PI-3K inhibitor prodrug for phase II clinical trials in the treatment of RCC.

A macrophage-dominant PI3K isoform controls hypoxia-induced HIF1α and HIF2α stability and tumor growth, angiogenesis, and metastasis.

UNLABELLED: 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 HIFα subunit and a constitutively expressed HIFß subunit. The signaling pathways operational in macrophages regulating hypoxia-induced HIFα stabilization remain the subject of intense investigation. Here, it was discovered that the PTEN/PI3K/AKT signaling axis controls hypoxia-induced HIF1α (HIF1A) and HIF2α (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 HIFα protein and its primary transcriptional target VEGF in response to hypoxia. Moreover, blocking the PI3K/AKT signaling axis promoted the hypoxic degradation of HIFα via the 26S proteasome. Mechanistically, a macrophage-dominant PI3K isoform (p110γ) directed tumor growth, angiogenesis, metastasis, and the HIFα/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 HIFα under hypoxic conditions, a signaling pathway in macrophages that controls tumor-induced angiogenesis and metastasis. IMPLICATIONS: This study indicates that PI3K inhibitors are excellent candidates for the treatment of cancers where macrophages promote tumor progression.

Rac2 controls tumor growth, metastasis and M1-M2 macrophage differentiation in vivo.

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 α4ß1 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 α4ß1 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.