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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.

Supplementary Figure Legend from A Macrophage-Dominant PI3K Isoform Controls Hypoxia-Induced HIF1α and HIF2α Stability and Tumor Growth, Angiogenesis, and Metastasis

SF1126 and Sorafenib inhibit cell cycle progression by inducing apoptosis

File contains Supplementary Methods and References

Contain description of Supplementary methods and legends to supplementary figures and tables

Supplementary Fig. S1 shows JQ1 blocks IL4 induced CD206 expression. Supplementary Fig. S2 shows Differentially expressed genes in LPS-stimulated or IL4 stimulated BMDMs. Supplementary Fig. S3 shows Effect of JQ1 on LLC tumor growth. Supplementary Fig. S4 shows SF1126 blocks tumor growth and immunosuppressive macrophage polarization. Supplementary Fig. S5 shows JQ1 and SF2523 reduce tumor growth due to its effect on macrophages. Supplementary Fig. S6 shows SF2523 inhibits B16 tumor metastasis. Supplementary Table S1 shows Primers used for ChIP analysis. Supplementary Table S2 shows Contingency tables representing the effects on the number of shared over or under expressed genes of different exposures.

Chau Talalay Synergy studies on HCC cells

Supplementary Figures 1-5 from A Vascular Targeted Pan Phosphoinositide 3-Kinase Inhibitor Prodrug, SF1126, with Antitumor and Antiangiogenic Activity

Hypoxia and necrosis are fundamental features of glioblastoma (GBM) and their emergence is critical for the rapid biological progression of this fatal tumor; yet, underlying mechanisms are poorly understood. We have suggested that vaso-occlusion following intravascular thrombosis could initiate or propagate hypoxia and necrosis in GBM. Tissue factor (TF), the main cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Epidermal growth factor receptor (EGFR) amplification and PTEN loss are two common genetic alterations seen in GBM but not in lower-grade astrocytomas that could be responsible for TF up-regulation. The most frequent EGFR mutation in GBM involves deletion of exons 2 to 7, resulting in the expression of a constitutively active receptor, EGFRvIII. Here, we show that overexpression of EGFR or EGFRvIII in human glioma cells causes increased basal TF expression and that stimulation of EGFR by its ligand, EGF, leads to a marked dose-dependent up-regulation of TF. In all cases, increased TF expression led to accelerated plasma coagulation in vitro. EGFR-mediated TF expression depended most strongly on activator protein-1 (AP-1) transcriptional activity and was associated with c-Jun NH2-terminal kinase (JNK) and JunD activation. Restoration of PTEN expression in PTEN-deficient GBM cells diminished EGFR-induced TF expression by inhibiting JunD/AP-1 transcriptional activity. PTEN mediated this effect by antagonizing phosphatidylinositol 3-kinase activity, which in turn attenuated both Akt and JNK activities. These mechanisms are likely at work in vivo, as EGFR expression was highly correlated with TF expression in human high-grade astrocytoma specimens. [Cancer Res 2009;69(6):2540–9]

Supplementary Figures and Tables from Epidermal Growth Factor Receptor and PTEN Modulate Tissue Factor Expression in Glioblastoma through JunD/Activator Protein-1 Transcriptional Activity

PTEN and the pan phosphoinositide 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one (LY294002) exert significant control over tumor-induced angiogenesis and tumor growth in vivo. The LY294002 compound is not a viable drug candidate due to poor pharmacologic variables of insolubility and short half-life. Herein, we describe the development and antitumor activity of a novel RGDS-conjugated LY294002 prodrug, termed SF1126, which is designed to exhibit increased solubility and bind to specific integrins within the tumor compartment, resulting in enhanced delivery of the active compound to the tumor vasculature and tumor. SF1126 is water soluble, has favorable pharmacokinetics, and is well tolerated in murine systems. The capacity of SF1126 to inhibit U87MG and PC3 tumor growth was enhanced by the RGDS integrin (αvβ3/α5β1) binding component, exhibiting increased activity compared with a false RADS-targeted prodrug, SF1326. Antitumor activity of SF1126 was associated with the pharmacokinetic accumulation of SF1126 in tumor tissue and the pharmacodynamic knockdown of phosphorylated AKT in vivo. Furthermore, SF1126 seems to exhibit both antitumor and antiangiogenic activity. The results support SF1126 as a viable pan PI3K inhibitor for phase I clinical trials in cancer and provide support for a new paradigm, the application of pan PI3K inhibitory prodrugs for the treatment of cancer. [Cancer Res 2008;68(1):206–15]

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.

Emerging new variants of SARS-CoV-2 and inevitable acquired drug resistance call for the continued search of new pharmacological targets to fight the potentially fatal infection. Here, we describe the mechanisms by which the E protein of SARS-CoV-2 hijacks the human transcriptional regulator BRD4. We found that SARS-CoV-2 E is acetylated in vivo and co-immunoprecipitates with BRD4 in human cells. Bromodomains (BDs) of BRD4 bind to the C-terminus of the E protein, acetylated by human acetyltransferase p300, whereas the ET domain of BRD4 recognizes the unmodified motif of the E protein. Inhibitors of BRD4 BDs, JQ1 or OTX015, decrease SARS-CoV-2 infectivity in lung bronchial epithelial cells, indicating that the acetyllysine binding function of BDs is necessary for the virus fitness and that BRD4 represents a potential anti-COVID-19 target. Our findings provide insight into molecular mechanisms that contribute to SARS-CoV-2 pathogenesis and shed light on a new strategy to block SARS-CoV-2 infection.

Summary Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma and one of the most challenging blood cancers to combat due to frequent relapse after treatment. Here, we developed the first-in-class BTK/PI3K/BRD4 axis inhibitor SRX3262, which simultaneously blocks three interrelated MCL driver pathways – BTK, PI3K-AKT-mTOR and MYC. SRX3262 concomitantly binds to BTK, PI3K, and BRD4, exhibits potent in vitro and in vivo activity against MCL, and overcomes the Ibrutinib resistance resulting from the BTK-C481S mutation. Our results reveal that SRX3262 inhibits IgM-induced BTK and AKT phosphorylation and abrogates binding of BRD4 to MYC loci. SRX3262 promotes c-MYC destabilization, induces cell cycle arrest and apoptosis, and shows antitumor activity in in vivo xenograft models. Together, our study provides mechanistic insights and rationale for the use of the triple BTK/PI3K/BRD4 activity inhibitors as a new approach to treat MCL., Graphical abstract, Highlights • BTK/PI3K/BRD4 axis inhibitor SRX3262 simultaneously blocks three MCL driver pathways • SRX3262 binds to BTK, PI3K, and BRD4 and exhibits potent anti-MCL activity in vivo • SRX3262 inhibits BTK and AKT phosphorylation and abrogates binding of BRD4 to MYC • SRX3262 promotes c-MYC destabilization and induces cell cycle arrest and apoptosis, Medical biochemistry; Molecular physiology; Cancer

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.

Pathogenic viruses like SARS-CoV-2 and HIV hijack the host molecular machinery to establish infection and survival in infected cells. This has led the scientific community to explore the molecular mechanisms by which SARS-CoV-2 infects host cells, establishes productive infection, and causes life-threatening pathophysiology. Very few targeted therapeutics for COVID-19 currently exist, such as remdesivir. Recently, a proteomic approach explored the interactions of 26 of 29 SARS-CoV-2 proteins with cellular targets in human cells and identified 67 interactions as potential targets for drug development. Two of the critical targets, the bromodomain and extra-terminal domain proteins (BETs): BRD2/BRD4 and mTOR, are inhibited by the dual inhibitory small molecule SF2523 at nanomolar potency. SF2523 is the only known mTOR PI3K-α/(BRD2/BRD4) inhibitor with potential to block two orthogonal pathways necessary for SARS-CoV-2 pathogenesis in human cells. Our results demonstrate that SF2523 effectively blocks SARS-CoV-2 replication in lung bronchial epithelial cells in vitro, showing an IC50 value of 1.5 µM, comparable to IC50 value of remdesivir (1.1 µM). Further, we demonstrated that the combination of doses of SF2523 and remdesivir is highly synergistic: it allows for the reduction of doses of SF2523 and remdesivir by 25-fold and 4-fold, respectively, to achieve the same potency observed for a single inhibitor. Because SF2523 inhibits two SARS-CoV-2 driven pathogenesis mechanisms involving BRD2/BRD4 and mTOR signaling, our data suggest that SF2523 alone or in combination with remdesivir could be a novel and efficient therapeutic strategy to block SARS-CoV-2 infection and hence be beneficial in preventing severe COVID-19 disease evolution.One Sentence SummaryEvidence of in silico designed chemotype (SF2523) targeting PI3K-α/mTOR/BRD4 inhibits SARS-CoV-2 infection and is highly synergistic with remdesivir.

The PI3K-AKT-mTOR pathway has been at the center of anti-cancer drug development1,2, but targeted inhibition of PI3K kinase activity is proven to provide a limited therapeutic effect and is often followed by the development of resistance to the drug3. This limitation could in part be alleviated through concurrently inhibiting multiple pathological pathways characterized by synthetic lethality relationship. For example, beneficial effects in metastatic breast cancer are observed when inhibition of PI3K is combined with BET proteins inhibition4. While the need for combinations of single agent targeted therapies has become clear, this approach commonly requires dose reduction of the individual agents due to additive toxicity that may compromise efficacy5., This work was supported by the NIH grants CA192656 (D.L.D.), FD005113 (D.L.D., A.M.B.), GM135671 (T.G.K.), GM125195 (T.G.K.) and the Tower Cancer Research Foundation grant (A.M.B.). K.R.V. is a CFReT Fellow.

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.

Venetoclax (VEN), a BCL2 specific inhibitor, has shown excellent clinical activities in various types of non-Hodgkin lymphoma, and it is FDA-approved in patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). However, its efficacy has been mostly disappointing in BCL2-positive (BCL2+) lymphomas harboring classic BCL2 rearrangements, including follicular lymphoma (FL), double-hit lymphoma (DHL), and triple-hit lymphoma (THL). The mechanism by which BCL2+ lymphomas evade BCL2 inhibition remains elusive although it has shown to be in part due to overexpression of anti-apoptotic proteins like BCLxL or MCL1. Aurora kinases (AURK) are serine threonine kinases involved in mitotic regulation and have functional role in stabilization of regulatory proteins such as MYC. Here in we investigated potential mechanisms of primary resistance to VEN and the effect of AURK inhibition in overcoming primary VEN failure in BCL2+ lymphomas. BCL2+ lymphoma cells, WSU-NHL (single hit; BCL2 only), DoHH2 (DHL; BCL2 and MYC), and VAL (THL; BCL2, MYC, and BCL6) were evaluated for cell viability (ATP quantification) and apoptosis (Annexin V/7AAD staining), after treatment with various concentrations of VEN with or without MLN8237 (AURK-A inhibitor), LY3295668 (AURK-A inhibitor), or AZD2811 (AURK-B inhibitor). Addition of an AURK-A or B inhibitor to VEN induced robust killing and displayed synergism only in BCL2+ but not in BCL2-negative Raji and Ramos Burkitt lymphoma cells (MYC only). AURK-A inhibition using MLN8237 was chosen for further in-depth functional analysis. Immunoblotting revealed increased caspase-3 cleavage in DoHH2 cells treated with VEN+MLN8237 combination than either agent alone. No significant changes in BCL2, BCLxL or MCL1 protein levels were noticed in DoHH2 and VAL cells after single or combined treatments. However, MLN8237 resulted in elevated levels of proapoptotic proteins BAX and PUMA. MYC degradation occurred later in cells after treatment with MLN8237 or combination implying that MYC degradation may be a delayed and independent effect. Furthermore, VEN+MLN8237 combination completely cleared tumors in two different BCL2+ lymphoma mouse models where mice were randomized into four groups and treated with vehicle, VEN, MLN8237, or VEN+MLN8237 combination via oral gavage for 15 days. First, in a DoHH2 DHL xenograft SCID mouse model, VEN+MLN8237 combination resulted in complete tumor regression and 100% tumor-free survival on day 100 (p < 0.0001; N=8/group) with no discernable toxicity, while all mice in other groups were euthanized due to disease progression within 45 days. Next, in a disseminated THL model using VAL cells intravenously infused into NCG mice, all animals receiving combination therapy survived with no evidence of disease on day 100 (p < 0.0001; N=6-8/group), while all except one in other groups were euthanized due to removal criteria, including hindlimb paralysis and weight loss, by day 60. To investigative the tumor response to BCL2 and AURK inhibitions, we performed transcriptome sequencing (RNA seq) of DoHH2 tumors harvested from SCID mice (N=6-7/group) treated for 3 days under the 4 conditions as described above. Comparison of VEN with VEN+MLN8237 combination identified 41 genes of which 33 increased and 8 decreased in combination therapy compared to VEN alone (Fold change >2 and FDR < 0.05). Most notably, CDKN1A (p21) level was decreased by 2-fold in VEN monotherapy compared to vehicle control while the concurrent inhibition of AURK-A by MLN8237 reversed this process by upregulating p21 by > 4-fold compared to VEN monotherapy. Ingenuity pathway analysis subsequently revealed that VEN+MLN8237 combination induced significant upregulation of p53/p21/BAX network. Additional assays confirmed an early characteristic downregulation of p53 protein levels in response to VEN treatment in BCL2+ lymphoma cells. The induction of p53, p21, PUMA, and BAX in VEN+MLN8237 combination was further confirmed by immunoblotting. In contrast, VEN reduced p21, PUMA and BAX expression levels compared to vehicle treated cells. p53 knockdown in DoHH2 cells resulted in similar resistance to VEN and combination treatment. Taken together these data suggest AURK inhibition overcomes downregulation of p53/p21/BAX axis by BCL2+ lymphomas in response to BCL2 inhibition, hence lay the groundwork for further evaluation of this combination in clinical settings. Figure 1 Figure 1. Disclosures Foureau: Cytognos: Honoraria; TeneoBio, Celgene: Research Funding. Ghosh: Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Epizyme: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Genmab: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; ADC Therapeutics: Consultancy, Honoraria; Adaptive Biotech: Consultancy, Honoraria; AbbVie: Honoraria, Speakers Bureau; Karyopharma: Consultancy, Honoraria; Genentech: Research Funding. Copelan: Amgen: Consultancy. Durden: SignalRx Pharmaceuticals: Current holder of individual stocks in a privately-held company. Avalos: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; BMJ Best Practice: Patents & Royalties: Royalties from a co-authored article on evaluation of neutropenia. Park: Takeda: Research Funding; Rafael Pharma: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Morphosys: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding, Speakers Bureau; Gilead: Speakers Bureau; G1 Therapeutics: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Mantle cell lymphoma (MCL) is an aggressive, rare, and difficult to treat subtype of non-Hodgkin's Lymphoma (NHL) that accounts for about 6% of all cases. Although there is no defined standard of care for MCL treatment, some combination of chemo-immunotherapy and rituximab maintenance with or without autologous stem cell transplantation is generally employed depending on the age and fitness of the patient. Despite recent development of novel therapeutics, there is inevitable disease relapse with progressively declining efficacy and increasing frequency of resistance with single agent targeted therapy. Here, we describe the novel multitarget inhibitor SRX3177 which simultaneously hits three oncogenic targets: phosphatidylinositol-3 kinase (PI3K), cyclin-dependent kinases 4 and 6 (CDK4/6), and the epigenetic reader protein BRD4. This in silico designed, thieno-pyranone (TP) scaffold-based small molecule inhibitor orthogonally disrupt three targets within the cancer cell with one agent. Targeting the cell cycle with small molecule inhibitors represents a reasonable attempt to treat MCL, as cell cycle-associated genes like ATM, TP53, CDKN2A, CCND1 and CDK4/6 are most frequently mutated in patients. Palbociclib is a well-known single agent CDK4/6 inhibitor that has been employed in both solid and hematological malignancies. Due to its cytostatic nature, treatment with single agent palbociclib often results in the emergence of treatment-resistant clones. Therefore, a combination strategy would theoretically be more effective and can overcome the development of resistance. Moreover, prolonged G1 arrest by CDK4/6 inhibition sensitizes lymphoma cells to PI3K inhibition, suggesting a synthetic lethality relationship between these two agents. Inhibiting the chromatin reader protein BRD4 causes downregulation of target genes c-MYC and BCL2, further increasing cytotoxic capabilities. Hence, we developed SRX3177 as a potent CDK4/6/PI3K/BRD4 triple inhibitor to synergistically inhibit cell cycle progression and induce cancer cell apoptosis. SRX3177 is an ATP competitive CDK4/6 inhibitor (IC 50: CDK4 = 2.54 nM, CDK6 = 3.26 nM), PI3K inhibitor (IC 50: PI3Kα = 79.3 nM, PI3Kδ = 83.4 nM), and BRD4 inhibitor (IC 50: BD1 = 32.9 nM, BD2 = 88.8 nM). We have tested the efficacy of SRX3177 against a panel of MCL cell lines and report that SRX3177 induces a strong antiproliferative activity with maximal IC 50 0f 340 nM in JeKo-1, 29 nM in Mino cells, and 630 nM for Rec-1 cells while IC 50 values for cell lines Granta and JVM-2 were 1.3 µM and 1.5 µM, respectively. Further, we show that SRX3177 is more potent to tumor cells than the individual PI3K (BKM120), BTK (Ibrutinib), BRD4 (JQ1), and CDK4/6 (palbociclib) inhibitors, and dual PI3K/BRD4 inhibitor SF2523 (backbone for SRX3177) in JeKo-1 cells. Next, we examine the cytotoxic effect of SRX3177 in ibrutinib/palbociclib resistant primary MCL cells. Our results show that SRX3177 triggers cytotoxic response at 500 nM and 1000 nM as compared to the lack of cytotoxicity of combination Ibrutinib and palbociclib at 150 nM and 1000 nM (Fig 1). SRX3177 induces a strong apoptotic response and cell cycle arrest in JeKo-1 and Mino cells at 24hrs. Annexin V/7AAD apoptosis staining confirmed the induction of PCD by SRX3177with increase in c-PARP. Western blot analysis shows SRX3177 treatment blocks both PI3K/AKT signaling and Rb phosphorylation. Moreover, analysis by chromatin immunoprecipitation revealed that SRX3177 effectively blocked BRD4 binding to both the promoter and enhancer of c-MYC (p≤0.01 and p≤0.001) and BCL2 (p≤0.05). SRX3177 also suppresses the c-MYC and BCL2 transcriptional program in both a time- and dose-dependent manner. Our findings also demonstrate a SRX3177-dependent reduction in c-MYC half-life via induction of proteasomal-mediated degradation. This degradation is associated with decreased phosphorylation of c-MYC at Ser62 and increased phosphorylation of c-MYC at Thr58 - indicative of differential regulation of c-MYC stability. Finally, we show that SRX3177 overcomes chronic ibrutinib resistance in Jeko-1 cells with a maximal IC 50 of 150 nM as compared to 64 µM with ibrutinib. Hence, the triple inhibitor SRX3177 has superior potency to ibrutinib in MCL cell lines and succeeds in overcoming ibrutinib-resistance at nanomolar doses. Taken together, our data supports the development of SRX3177 as a novel therapeutic agent for treatment of MCL. Figure 1 Figure 1. Disclosures Martin: ADCT: Consultancy. Park: Takeda: Research Funding; G1 Therapeutics: Consultancy; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding, Speakers Bureau; Gilead: Speakers Bureau; Rafael Pharma: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Morphosys: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding. Durden: SignalRx Pharmaceuticals: Current holder of individual stocks in a privately-held company.

Bone-metastatic castration-resistant prostate cancer (CRPC) is lethal due to inherent resistance to androgen deprivation therapy, chemotherapy, and targeted therapies. Despite the fact that a majority of CRPC patients (approximately 70%) harbor a constitutively active PI3K survival pathway, targeting the PI3K/mTOR pathway has failed to increase overall survival in clinical trials. Here, we identified two separate and independent survival pathways induced by the bone tumor microenvironment that are hyperactivated in CRPC and confer resistance to PI3K inhibitors. The first pathway involves integrin α6β1-mediated adhesion to laminin and the second involves hypoxia-induced expression of PIM kinases. In vitro and in vivo models demonstrate that these pathways transduce parallel but independent signals that promote survival by reducing oxidative stress and preventing cell death. We further demonstrate that both pathways drive resistance to PI3K inhibitors in PTEN-negative tumors. These results provide preclinical evidence that combined inhibition of integrin α6β1 and PIM kinase in CRPC is required to overcome tumor microenvironment-mediated resistance to PI3K inhibitors in PTEN-negative tumors within the hypoxic and laminin-rich bone microenvironment.

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.

Neuroblastoma are pediatric, extracranial malignancies showing alarming survival prognosis outcomes due to their resilience to current aggressive treatment regimens, including chemotherapies with cisplatin (CDDP) provided in the first line of therapy regimens. Metabolic deregulation supports tumor cell survival in drug-treated conditions. However, metabolic pathways underlying cisplatin-resistance are least studied in neuroblastoma. Our metabolomics analysis revealed that cisplatin-insensitive cells alter their metabolism, especially, the metabolism of amino acids was upregulated in cisplatin-insensitive cells compared to the cisplatin-sensitive neuroblastoma cell line. A significant increase in amino acid levels in cisplatin-insensitive cells led us to hypothesize that the mechanisms upregulating intracellular amino acid pools facilitate insensitivity in neuroblastoma. We hereby report that amino acid depletion reduces cell survival and cisplatin-insensitivity in neuroblastoma cells. Since cells regulate their amino acids levels through processes, such as autophagy, we evaluated the effects of hydroxychloroquine (HCQ), a terminal autophagy inhibitor, on the survival and amino acid metabolism of cisplatin-insensitive neuroblastoma cells. Our results demonstrate that combining HCQ with CDDP abrogated the amino acid metabolism in cisplatin-insensitive cells and sensitized neuroblastoma cells to sub-lethal doses of cisplatin. Our results suggest that targeting of amino acid replenishing mechanisms could be considered as a potential approach in developing combination therapies for treating neuroblastomas.

Cancer immunotherapy, including immune checkpoint blockade and adoptive CAR T-cell therapy, has clearly established itself as an important modality to treat melanoma and other malignancies. Despite the tremendous clinical success of immunotherapy over other cancer treatments, this approach has shown substantial benefit to only some of the patients while the rest of the patients have not responded due to immune evasion. In recent years, a combination of cancer immunotherapy together with existing anticancer treatments has gained significant attention and has been extensively investigated in preclinical or clinical studies. In this review, we discuss the therapeutic potential of novel regimens combining immune checkpoint inhibitors with therapeutic interventions that (1) increase tumor immunogenicity such as chemotherapy, radiotherapy, and epigenetic therapy; (2) reverse tumor immunosuppression such as TAMs, MDSCs, and Tregs targeted therapy; and (3) reduce tumor burden and increase the immune effector response with rationally designed dual or triple inhibitory chemotypes.

Vascular remodelling is a prominent feature of haemophilic arthropathy (HA) that may underlie re-bleeding, yet the nature of vascular changes and underlying mechanisms remain largely unknown. Here, we aimed to characterize synovial vascular remodelling and vessel integrity after haemarthrosis, as well as temporal changes in inflammatory and tissue-reparative pathways. Thirty acutely painful joints in patients with haemophilia (PWH) were imaged by musculoskeletal ultrasound with Power Doppler (MSKUS/PD) to detect vascular abnormalities and bloody effusions. Nineteen out of 30 painful joint episodes in PWH were associated with haemarthrosis, and abnormal vascular perfusion was unique to bleeding joints. A model of induced haemarthrosis in factor VIII (FVIII)-deficient mice was used for histological assessment of vascular remodelling (α-smooth muscle actin [αSMA] expression), and monitoring of in vivo vascular perfusion and permeability by MSKUS/PD and albumin extravasation, respectively. Inflammatory (M1) and reparative (M2) macrophage markers were quantified in murine synovium over a 10-week time course by real-time polymerase chain reaction. The abnormal vascular perfusion observed in PWH was recapitulated in FVIII-deficient mice after induced haemarthrosis. Neovascularization and increased vessel permeability were apparent 2 weeks post-bleed in FVIII-deficient mice, after a transient elevation of inflammatory macrophage M1 markers. These vascular changes subsided by week 4, while vascular remodelling, evidenced by architectural changes and pronounced αSMA expression, persisted alongside a reparative macrophage M2 response. In conclusion, haemarthrosis leads to transient inflammation coupled with neovascularization and associated vascular permeability, while subsequent tissue repair mechanisms coincide with vascular remodelling. Together, these vascular changes may promote re-bleeding and HA progression.

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.

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.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.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.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.

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 α4β1 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.

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α-HIF1β 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.

Low dose Poly-ICLC (Polyinosinic-Polycytidylic acid stabilized with polylysine and carboxymethylcellulose) has a direct immune enhancing action independent of interferon, including increased antibody response to antigen, and NK cell, T-cell, macrophage and cytokine activation. METHODS: We conducted an immunotherapy trial to evaluate the effect of Poly-ICLC in the children with recurrent or progressive low grade gliomas. Criteria for enrollment: progression by MRI and or visual deterioration. Patients received Poly-ICLC 20mcg/kg/dose IM injection twice a week for up to 24 months. RESULTS: 23 patients enrolled, 22 evaluable. Ages 2-20 years. Ten females and 13 males Location: suprasellar (11), spine (1), disseminated (4), others (7). Six patients with NF-1. Pathology: JPA (11), pilomyxoid (5), no pathology (3), Diffuse Astrocytoma, ganglioglioma, Glioneuronal tumor, LGG NOS (1 each). Follow-up range 3 to 58 months. Responses: 43% stable disease, 17% (4/23) partial responses- 3 with NF-1. Visual fields and acuity improved in 33% of patients with NF-1. Two JPA patients that have completed 24 months of therapy: one non-NF with SD and PFS 58 months, one NF-1, PR, PFS for 28 months. Most common toxicities included: fevers, discomfort at the injection site, Grade 1 neutropenia, lymphocytopenia, hypophosphatemia, ALT elevation. One case of grade 4 intratumoral hemorrhage, required medical management alone. No patient required transfusion or admission for fever and neutropenia. No reports of neuropathy. CONCLUSION: Poly-ICLC is well tolerated, minimal toxicity, radiographic and clinical responses were demonstrated in patients with LGG. Larger clinical trials with Poly-ICLC are being designed.

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.

Many side effects of current FDA-approved L-asparaginases have been related to their secondary L-glutaminase activity. The Wolinella succinogenes L-asparaginase (WoA) has been reported to be L-glutaminase free, suggesting it would have fewer side effects. Unexpectedly, the WoA variant with a proline at position 121 (WoA-P121) was found to have L-glutaminase activity in contrast to Uniprot entry P50286 (WoA-S121) that has a serine residue at this position. Towards understanding how this residue impacts the L-glutaminase property, kinetic analysis was coupled with crystal structure determination of these WoA variants. WoA-S121 was confirmed to have much lower L-glutaminase activity than WoA-P121, yet both showed comparable L-asparaginase activity. Structures of the WoA variants in complex with L-aspartic acid versus L-glutamic acid provide insights into their differential substrate selectivity. Structural analysis suggests a mechanism by which residue 121 impacts the conformation of the conserved tyrosine 27, a component of the catalytically-important flexible N-terminal loop. Surprisingly, we could fully model this loop in either its open or closed conformations, revealing the roles of specific residues of an evolutionary conserved motif among this L-asparaginase family. Together, this work showcases critical residues that influence the ability of the flexible N-terminal loop for adopting its active conformation, thereby effecting substrate specificity.

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.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvβ3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvβ3 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 αvβ3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvβ3 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 αvβ3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvβ3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma.

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.

SF1126 is a peptidic pro-drug inhibitor of pan-PI3K/mTORC. A first-in-human study evaluated safety, dose limiting toxicities (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of SF1126, in patients with advanced solid and B-cell malignancies.SF1126 was administered IV days 1 and 4, weekly in 28day-cycles. Dose escalation utilised modified Fibonacci 3+3. Samples to monitor PK and PD were obtained.Forty four patients were treated at 9 dose levels (90-1110 mg/m(2)/day). Most toxicity was grade 1 and 2 with a single DLT at180 mg/m(2) (diarrhoea). Exposure measured by peak concentration (C(max)) and area under the time-concentration curve (AUC(0-)(t)) was dose proportional. Stable disease (SD) was the best response in 19 of 33 (58%) evaluable patients. MTD was not reached but the maximum administered dose (MAD) was 1110 mg/m(2). The protocol was amended to enrol patients with CD20+ B-cell malignancies at 1110 mg/m(2). A CLL patient who progressed on rituximab [R] achieved SD after 2 months on SF1126 alone but in combination with R achieved a 55% decrease in absolute lymphocyte count and a lymph node response. PD studies of CLL cells demonstrated SF1126 reduced p-AKT and increased apoptosis indicating inhibition of activated PI3K signalling.SF1126 is well tolerated with SD as the best response in patients with advanced malignancies.

Introduction: Acute lymphoblastic leukemia is the most prevalent form of cancer affecting children with 2,500-3,500 new cases per year. More effective targeted therapies have yielded a current five-year survival rate of at least 85% for children; however relapsed disease, as well as harsh side effects of treatment, remain prevalent hurdles for many patients. Cells harbored by the bone marrow comprise minimal residual disease that may contribute to later re-expansion of the tumor population following treatment, also known as cell adhesion mediated drug resistance (CAM-DR). Bone marrow stromal cell contact has been shown to upregulate phosphorylated AKT, promoting survival of ALL cells. We investigate a new dual targeted therapy, Morpholinothienopyrane (SF2535), which inhibits both PI3Kdelta and BRD4, each key molecules in signal transduction pathways between microenvironment and leukemia cells. PI3Kdelta is a central nodal molecule in outside-in pathways including cell-cell interactions mediated by integrins. BRD4, a member of the bromodomain and extraterminal domain (BET) family of proteins which bind acetylated lysines at promoter and enhancer regions, is another key player involved in regulation of transcription of myc family transcription factors. In addition to transcriptional regulation by BRD4, myc is also regulated by PI3kdelta which inhibits GSK3beta-mediated degradation of myc. We hypothesize that this dual inhibitor, SF2535, will disrupt signaling between leukemia cells and the bone marrow microenvironment, thus addressing CAM-DR. Methods/Results: In vitro and in vivo BRD4 and PI3Kdelta target downregulation was measured by western blot including assessment of myc, AKT, and phosphorylated AKT. Cells were starved in vitro in serum-free conditions for 18 hours followed by treatment with SF2535 and stimulation with serum. Levels of phosphorylated AKT were noticeably decreased in SF2535 treated cells compared with DMSO control or JQ1 (BET inhibitor) treatment alone. Truncation of microenvironmental outside-in signaling via inhibition of PI3Kdelta and BRD4 was measured via transwell migration assay. Primary ALL cells were cultured on top of a porous membrane with SDF-1alpha in the chamber below. Significantly less primary ALL cells migrated toward SDF-1alpha when treated with SF2535 compared to DMSO, CAL101 (PI3Kdelta inhibitor), or JQ1 (BET inhibitor). The combination of SF2535 with vincristine, dexamethasone, and L-asparaginase (VDL), the backbone of induction therapy for many ALL patients, was evaluated in vitro. Primary tumor cells were co-cultured on irradiated OP-9 murine stromal cells and treated with SF2535, VDL, or SF2535 and VDL combined. Apoptosis in response to treatment was measured via AnnexinV and 7-AAD flow cytometry. The combination of SF2535 and VDL showed significantly more cell death compared to either SF2535 or VDL alone at three concentrations of each treatment. Additionally, CalcuSyn software was used to assess putative additive versus synergistic effects of this combination. CalcuSyn analysis of VDL and SF2535 combination compared to either SF2535 or VDL treatment alone shows synergy (defined as a combination index of 0.3-0.7) between these two therapies used on sample LAX7R after 1 day of treatment. Discussion: Thus far we have shown that this novel inhibitor decreases expression of downstream targets of PI3Kdelta (phosphorylated AKT) and BRD4 (C-myc), inhibits migration of ALL toward bone marrow stromal factors, and increases apoptosis of primary patient samples when combined with VDL compared to VDL alone. Most notably, we have determined a synergistic relationship between SF2535 and VDL in vitro, suggesting a role for this novel therapy in sensitizing cells to chemotherapy. The use of a single agent against both PI3Kdelta and BRD4 ultimately aimed at downregulation of myc levels provides the possibility of more effective, less toxic therapeutic option that addresses CAM-DR faced by many patients. While ongoing experiments in vitro and in vivo will provide a more robust picture of the efficacy of this drug, our preliminary data suggest promise for this novel application of BRD4 and PI3Kdelta inhibition in the context of acute lymphoblastic leukemia. Disclosures Durden: SignalRx Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

BACKGROUND: Polyinosinic-Polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC) is a double stranded RNA that acts as a ligand for the Toll Receptor 3 (TLR3) and results in a broad host defense stimulation, including T-cell and natural killer cell activation and cytokine release (interferons alpha, beta, and gamma, interleukins, corticosteroids, and TNF). The goal of this trial is to determine the efficacy of this novel immunotherapy against refractory pediatric low-grade glioma (LGG). Here we present the results for the neurofibromatosis group. METHODS: Poly-ICLC 20mcg/kg/dose IM was self-administered twice a week at home for up to 24 cycles. Each cycle is 28 days. Endpoints include objective tumor response rate, progression-free survival (PFS), and rate of stabilization/improvement in visual examination in those with optic pathway glioma. RESULTS: Twenty three patients were enrolled, six with NF-1. Of NF1 patients (3 males, 3 females), five were evaluable, ages 1–16 years. Tumor location: 4 optic pathway, one thalamic, one cervico-medullary junction. 5/6 had received more than 2 prior regimens (range 1–7). Responses (PR+SD) were observed in 4/5 (80%), with 2 SD having a decrease in tumor size by 35% and 10%, one of whom had significant improvement on visual field testing, and 2 PR having decrease in tumor size by 50% and 55%. Only low grade toxicities were observed with poly-ICLC, including erythema and pain at site of injection, fever, myalgias, and ALT elevation. One intra-tumoral hemorrhage occurred with complete resolution with medical management alone. All patients are alive. CONCLUSION: Poly-ICLC is extremely well tolerated in the NF-1 group and the single agent response rate in this small NF1 cohort is encouraging. Expanded phase II testing of poly-ICLC in the NF-1 cohort is planned.

Purpose The purpose of the current study is to determine the in vitro cytotoxic effects of the novel pan-PI3-kinase inhibitor SF1126 in HER2-over-expressing breast cancer cells. Methods Cell proliferation and cytotoxicity were examined by MTS colorimetric assay, FACS analysis, colony formation assay, and immunoblotting. Phosphoinositol-3-kinase signaling was assessed by immunoblotting for phosphorylated Akt. Combination effects of trastuzumab and SF1126 were examined in resistant cells by MTS and soft agar assay. Results SF1126 inhibited proliferation, and induced G1 arrest and apoptosis of SKBR3 and BT474 parental and trastuzumab-resistant HER2-over-expressing cells. Colony formation was inhibited by SF1126, caspase 3 and PARP proteins were cleaved, and survivin was down-regulated. Inhibition of PI3-kinase was confirmed by reduced phosphorylation of Akt. Finally, the combination of SF1126 and trastuzumab synergistically inhibited proliferation of resistant cells, with SF1126-treated cells showing reduced anchorage-independent growth. Conclusions These results provide evidence that a clinically relevant pan-PI-3 kinase inhibitor can reverse trastuzumab resistance in breast cancer cells, and support further study of PI3-kinase inhibitor SF1126 in HER2-over-expressing breast cancer cells, including those that have progressed on trastuzumab.

Angiogenesis is tightly regulated by opposing mechanisms in mammalian cells and is controlled by the angiogenic switch. Other review articles have described a central role for the PTEN/PI-3 kinase/AKT signaling node in the coordinate control of cell division, tumor growth, apoptosis, invasion and cellular metabolism [1, 2]. In this review, we focus on literature that supports the PTEN/PI-3 kinase/AKT signaling node as a major control point for the angiogenic switch in both the on and off positions. We also discuss the rationale for designing small molecule drugs that target the PTEN/PI-3 kinase/AKT signaling node for therapeutic intervention. Our hypothesis is that, instead of inhibiting one cell surface receptor, such as VEGFR2 with bevacizumab (Avastin®), thereby leaving a significant number of receptors free to pulse angiogenic signals, a more effective strategy may be to regulate signaling through an intercept node where redundant cell surface receptor signals converge to transmit important signaling events within the cell. This therapeutic configuration brings coordinate control over multiple cell surface receptors in concert with a physiologic response which may combine arrest of cell cycle progression with growth inhibition and the induction of genes involved in specialized functions such as movement, which are all required for the complex process of angiogenesis to occur in a temporal-spatial paradigm.

Herein, we describe an obligate role for the hematopoietic specific GTPase, RAC2 in endothelial integrin signaling and the postnatal neovascularization response in vivo. Using a Rac2 knockout mouse model, we discovered that despite the presence of both RAC1 and RAC2 protein in endothelial cells, RAC2 is obligately required for the postnatal neovascular response and alphavbeta3/ alpha4beta1/alpha5beta1 integrin-directed migration on vitronectin, H296 and CH271, fibronectin fragments, respectively. The molecular basis for RAC2 specificity was explored. A genetic analysis of Syk -/+ or Syk-/+;Rac2 -/+ mice revealed that SYK kinase is required for the integrin induced activation of RAC2. The analysis of endothelial cells from Rac2-/+ versus Syk-/+;Rac2-/+ mice provided genetic evidence that SYK-RAC2 signaling axis regulates integrin (alphavbeta3, alpha4beta1 and alpha5beta1) dependent migration. Our results provide evidence that a specific region of the nonreceptor protein tyrosine kinase, SYK, the B linker region containing Y342 and Y346 is required for SYK's regulation of RAC2 and integrin dependent migration. Moreover, the capacity of mice to vascularize the ischemic hindlimb following femoral artery ligation or matrigel plugs was markedly reduced in mice homozygous deficient for the Rac2 gene. These findings identify a novel signaling axis for the induction and potential modulation of postnatal angiogenesis.

PTEN and the pan phosphoinositide 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one (LY294002) exert significant control over tumor-induced angiogenesis and tumor growth in vivo. The LY294002 compound is not a viable drug candidate due to poor pharmacologic variables of insolubility and short half-life. Herein, we describe the development and antitumor activity of a novel RGDS-conjugated LY294002 prodrug, termed SF1126, which is designed to exhibit increased solubility and bind to specific integrins within the tumor compartment, resulting in enhanced delivery of the active compound to the tumor vasculature and tumor. SF1126 is water soluble, has favorable pharmacokinetics, and is well tolerated in murine systems. The capacity of SF1126 to inhibit U87MG and PC3 tumor growth was enhanced by the RGDS integrin (αvβ3/α5β1) binding component, exhibiting increased activity compared with a false RADS-targeted prodrug, SF1326. Antitumor activity of SF1126 was associated with the pharmacokinetic accumulation of SF1126 in tumor tissue and the pharmacodynamic knockdown of phosphorylated AKT in vivo. Furthermore, SF1126 seems to exhibit both antitumor and antiangiogenic activity. The results support SF1126 as a viable pan PI3K inhibitor for phase I clinical trials in cancer and provide support for a new paradigm, the application of pan PI3K inhibitory prodrugs for the treatment of cancer. [Cancer Res 2008;68(1):206–15]

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvβ3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvβ3 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 αvβ3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvβ3 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 αvβ3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvβ3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma., This work was supported by grant CA81403 from the National Institutes of Health and a grant from the Concern Foundation. This work was also supported in part by National Institutes of Health grants U01 CA70903, CA60104 and CA02649 to RCS, grant CA14089 to SG, grants CA94233, CA192656 and FD004385 to DLD, the Nautica Malibu Triathlon Fund, the Bogart Pediatric Cancer Research Program and the T.J. Martell Foundation for Leukemia, Cancer, and AIDS Research.

The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a−/− embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a −/− mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a−/− mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.