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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.

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.

Fcγ receptor–mediated phagocytosis is a complex process involving the activation of protein tyrosine kinases, events that are potentially down-regulated by protein tyrosine phosphatases. We used the J774A.1 macrophage cell line to examine the roles played by the protein tyrosine phosphatase SHP-1 in the negative regulation of Fcγ receptor–mediated phagocytosis. Stimulation with sensitized sheep red blood cells (sRBCs) induced tyrosine phosphorylation of CBL and association of CBL with CRKL. These events were completely or partially abrogated by PP1 or the heterologous expression of dominant-negative SYK, respectively. Heterologous expression of wild-type but not catalytically inactive SHP-1 also completely abrogated the phagocytosis of IgG-sensitized sRBCs. Most notably, overexpressed SHP-1 associates with CBL and this association led to CBL dephosphorylation, loss of the CBL-CRKL interaction, and the suppression of Rac activation. These data represent the first direct evidence that SHP-1 is involved in the regulation of Fcγ receptor–mediated phagocytosis and suggest that activating signals mediated by SRC family kinases SYK, CBL, phosphatidyl inositol-3 (PI-3) kinase, and Rac are directly opposed by inhibitory signals through SHP-1.

Cross-linking of Fc receptors for IgA, FcR (CD89), on monocytes/macrophages is known to enhance phagocytic activity and generation of oxygen free radicals. We provide evidence here that the FcR signals through the γ subunit of FcɛRI in U937 cells differentiated with interferon γ (IFNγ). Our results provide the first evidence that FcR-mediated signals modulate a multimolecular adaptor protein complex containing Grb2, Shc, SHIP, CrkL, Cbl, and SLP-76. Cross-linking of FcRI using anti-FcRI induces the phosphorylation of the γ subunit as detected by mobility retardation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Stimulation of FcRI induced the tyrosine phosphorylation of Shc and increased the association of Grb2 with Shc and CrkL. Grb2 associates constitutively with Sos, and the latter undergoes mobility shift upon FcRI stimulation. The complex adapter proteins, Cbl and SLP-76, are physically associated in myeloid cells and both proteins undergo tyrosine phosphorylation upon FcR stimulation. These data indicate that the stimulation of FcR results in the modulation of adaptor complexes containing tyrosine-phosphorylated Cbl, Shc, SHIP, Grb2, and Crkl. Experiments performed with the Src kinase inhibitor, PP1, provide the first evidence that Src kinase activation is required for FcRI-induced production of superoxide anions and provide insight into the mechanism for FcR-mediated activation of downstream oxidant signaling in myeloid cells.

Cross-linking of Fc receptors for IgA, FcalphaR (CD89), on monocytes/macrophages is known to enhance phagocytic activity and generation of oxygen free radicals. We provide evidence here that the FcalphaR signals through the gamma subunit of FcepsilonRI in U937 cells differentiated with interferon gamma (IFNgamma). Our results provide the first evidence that FcalphaR-mediated signals modulate a multimolecular adaptor protein complex containing Grb2, Shc, SHIP, CrkL, Cbl, and SLP-76. Cross-linking of FcalphaRI using anti-FcalphaRI induces the phosphorylation of the gamma subunit as detected by mobility retardation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Stimulation of FcalphaRI induced the tyrosine phosphorylation of Shc and increased the association of Grb2 with Shc and CrkL. Grb2 associates constitutively with Sos, and the latter undergoes mobility shift upon FcalphaRI stimulation. The complex adapter proteins, Cbl and SLP-76, are physically associated in myeloid cells and both proteins undergo tyrosine phosphorylation upon FcalphaR stimulation. These data indicate that the stimulation of FcalphaR results in the modulation of adaptor complexes containing tyrosine-phosphorylated Cbl, Shc, SHIP, Grb2, and Crkl. Experiments performed with the Src kinase inhibitor, PP1, provide the first evidence that Src kinase activation is required for FcalphaRI-induced production of superoxide anions and provide insight into the mechanism for FcalphaR-mediated activation of downstream oxidant signaling in myeloid cells.

SLP-76 and Cbl are complex adapter proteins that have the capacity to bind to smaller adapter proteins, such as Grb2, which subsequently binds the nucleotide exchange protein Sos in the transmission of intracellular signals. SLP-76, Cbl, Shc, and Grb2 have been implicated in immunoreceptor tyrosine-based activation motif (ITAM) signaling, leading to activation of Ras. However, their mechanism of action has not been determined. To date, there have been no reports of SLP-76 involvement in FcγRI-receptor signaling and no data exist for an interaction between Cbl, Shc, and SLP-76 in vivo. We provide evidence that SLP-76, Cbl, and Shc are tyrosine phosphorylated on FcγRI-receptor stimulation and are associated with the adapter protein Grb2 in γ-interferon–differentiated U937 cells (U937IF). The interactions between SLP-76 and Cbl and SLP-76 and Grb2 are present in resting U937IF cells. However, the interaction between SLP-76 and Grb2 becomes augmented twofold on FcγRI-receptor aggregation. Our results provide the first evidence for a phosphorylation-dependent interaction between SLP-76 and Shc, induced at least 10-fold on FcγRI receptor stimulation. Our data indicate that a significant portion of a multimolecular complex containing Cbl, SLP-76, Shc, and Grb2 is distinct from a trimolecular complex containing the Ras guanine nucleotide exchanger Sos, Shc, and Grb2. FcγRI-induced tyrosine phosphorylation of SLP-76, Cbl, Shc, and the highly induced SLP-76-Shc interaction provide the first evidence that SLP-76 and Cbl are involved in FcγRI signaling and suggest a functional significance for these interactions in FcγRI signal relay in the control of Ras in myeloid cells.© 1998 by The American Society of Hematology.

SLP-76 and Cbl are complex adapter proteins that have the capacity to bind to smaller adapter proteins, such as Grb2, which subsequently binds the nucleotide exchange protein Sos in the transmission of intracellular signals. SLP-76, Cbl, Shc, and Grb2 have been implicated in immunoreceptor tyrosine-based activation motif (ITAM) signaling, leading to activation of Ras. However, their mechanism of action has not been determined. To date, there have been no reports of SLP-76 involvement in FcγRI-receptor signaling and no data exist for an interaction between Cbl, Shc, and SLP-76 in vivo. We provide evidence that SLP-76, Cbl, and Shc are tyrosine phosphorylated on FcγRI-receptor stimulation and are associated with the adapter protein Grb2 in γ-interferon–differentiated U937 cells (U937IF). The interactions between SLP-76 and Cbl and SLP-76 and Grb2 are present in resting U937IF cells. However, the interaction between SLP-76 and Grb2 becomes augmented twofold on FcγRI-receptor aggregation. Our results provide the first evidence for a phosphorylation-dependent interaction between SLP-76 and Shc, induced at least 10-fold on FcγRI receptor stimulation. Our data indicate that a significant portion of a multimolecular complex containing Cbl, SLP-76, Shc, and Grb2 is distinct from a trimolecular complex containing the Ras guanine nucleotide exchanger Sos, Shc, and Grb2. FcγRI-induced tyrosine phosphorylation of SLP-76, Cbl, Shc, and the highly induced SLP-76-Shc interaction provide the first evidence that SLP-76 and Cbl are involved in FcγRI signaling and suggest a functional significance for these interactions in FcγRI signal relay in the control of Ras in myeloid cells.© 1998 by The American Society of Hematology.

In this report we show that gamma-interferon (IFN) induces the expression of the nonreceptor protein tyrosine kinase, p72syk, and that cross-linking the Fc gamma RI receptor in IFN-differentiated U937 cells (U937IF cells) results in the activation of syk kinase. We show that syk is tyrosine phosphorylated (12-fold increase) after Fc gamma RI cross-linking. In vitro kinase assays demonstrate that the specific kinase activity of syk increased eightfold after Fc gamma RI cross- linking. The activation of signal transduction through the Fc gamma RI receptor, as measured by the respiratory burst, is associated with the tyrosine phosphorylation and catalytic activation of the syk kinase. We show that syk coprecipitates with the gamma subunit of the Fc gamma RI, Fc gamma RI gamma. The data suggest that p72syk is involved in signal transduction through the Fc gamma RI receptor, involving the Fc gamma RI gamma subunit.

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. To better understand these mechanisms, we investigated the time course of synovial, vascular, stromal and cartilage changes in response to a single induced hemarthrosis in FVIII-deficient mice, compared the observed changes in vascular architecture to the changes in mouse models of rheumatoid arthritis (RA) and osteoarthritis (OA), and explored the extent to which these findings correlated with the development of hemophilic arthropathy in patients. Following a single induced hemarthrosis in the FVIII-deficient mouse, we found soft tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture in the injured joint. While soft tissue changes were rapidly reversible, and cartilage changes were mild and mostly reversible, ongoing structural changes of abnormal vascularity persisted for months. These vascular changes involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105) and vascular endothelial growth factor (VEGF), as well as altered joint perfusion as determined by in vivo imaging. Surprisingly, these vascular changes were also seen in uninjured joints, suggesting systemic mediation of the observed effects. Changes in vascular architecture and pronounced expression of α-SMA were unique to hemophilia, and were not found in joint tissue obtained from RA or OA mice or from tissue from patients with OA or RA. Since FVIII-deficient mice do not suffer from spontaneous joint bleeding, human studies needed to be designed to reveal if such vascular changes were associated with joint bleeding and joint deterioration. Therefore, to determine if vascular perfusion changes were also present in hemophilia patients and to what extent they were related with joint bleeding and the degree of arthropathy, a cohort of 26 adult patients with hemophilia was studied prospectively. Radiographic Pettersson Scores and clinical Hemophilia Joint Health Scores (HJHS) were assessed and joint bleed status and vascularity changes were investigated by dynamic in vivo imaging using power Doppler (PD) during high resolution musculoskeletal ultrasound examinations of 156 joints (both elbows, knees and ankles of each patient) at baseline and, subsequently of 10 joints during painful episodes. Subclinical joint bleeding was present in 41% of joints at baseline. Positive PD signals were more frequently present in joints with subclinical bleeding than in joints without (83% vs. 55%; p< 0.01), while the mean PD signal score was significantly higher in bleeding joints (3.1 vs. 1.5; p < 0.01). Importantly, joint bleed status was independently associated with PD signal score (odds ratio [OR] = 1.45 [95% CI: 1.13, 1.86, p = 0.0035]) and with Pettersson score (OR = 1.21 [95%CI: 1.03, 1.43, p = 0.0185]) after adjusting for HJHS. Acute bleeding episodes, irrespective of occurring in previously non-bleeding or in previously subclinically bleeding joints only happened in joints where vascularity changes were present at baseline. Moreover, during these episodes, mean PD signal increased significantly from 3.5 at baseline to 6.0 (maximum 9 per joint; p≤0.05). Therefore, in vivo imaging demonstrated that those vascular changes, first observed in the FVIII-deficient mouse, were significantly associated with bleeding and joint deterioration in hemophilia patients. In aggregate, our observations in hemophilic mice and patients provide strong evidence that abnormal blood vessel formation and dynamic vascular remodeling facilitate and perpetuate joint bleeding in response to local and/or systemic stimuli. While changes in vascularity appear to be an important link to repeated bleeding and progression of arthropathy, the interrelations of bleeding and vascular changes are complicated and will require further study. These findings not only provide new insights into the pathobiology of progression of hemophilic arthroapthy, but also open new avenues to study molecular targets for angiogenesis inhibition to prevent aberrant vessel formation, potentially increasing the effectiveness of clotting factor replacement therapy. Disclosures Von Drygalski: CSL Behring: Honoraria, Speakers Bureau; Novo Nordisk: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Hematherix Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Research Funding; Pfizer: Honoraria; Baxalta: Honoraria, Research Funding. Mosnier:Hematherix Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Backround: Dysregulation of the PI3K/Akt signal transduction pathway has been implicated in the development of a number of malignancies, including multiple myeloma (MM). This cellular signaling mechanism and its downstream targets (eg mTOR) regulate cell growth, proliferation and apoptosis. SF1126 (Semafore) is a water soluble prodrug of the pan-PI3K inhibitor, LY294002, whose anti-proliferative and pro-apoptotic activity has been well described in the literature. Preclinical studies using SF1126 in a variety of malignancies including glioma, prostate, non-small cell lung cancer, and breast cancer appear promising and have demonstrated profound antiangiogenic effects mediated through VEGF inhibition. Aim: To demonstrate in vitro anti-myeloma activity of SF1126, alone and in combination with dexamethasone, bortezomib, and melphalan and evaluate their effects on downstream targets of PI3K/Akt. Methods: MM cell lines (MM.1R, MM.1S, RPMI 8226) were treated with SF1126 (1–100uM), dexamethasone (5uM), bortezomib (5nM), melphalan (10uM) alone, and in combination. Growth inhibition following treatment was measured by MTT assay at 24 and 48 hours. Apoptosis was assessed by annexin-V binding assay using flow cytometry. Immunoblot analysis was performed to measure downstream targets of Akt including: p-PDK1 and mTOR (4E-BP1). Results: A clear dose response was established with an IC50 of 8.75uM in the MM.1R and 7.5uM in the MM.1S cell lines at 48 hours. At 24 and 48 hours, 5uM SF1126 alone resulted in 80% and 64% cell viability by MTT assay, respectively, in the MM.1R cell line. The combination of 5uM SF1126 with conventional agents was then tested in the MM.1R cell line. Combination with 5uM dexamethasone enhanced the efficacy of 5uM SF1126 by 26% at 48 hours. Combination with 10uM melphalan enhanced the efficacy of 5uM SF1126 by 20% at 24 hours. The combination with 5nM bortezomib enhanced the efficacy of 5uM SF1126 by 23% at 48 hours. Given prior experience demonstrating that short exposure to bortezomib activates Akt, we tested sequential administration of bortezomib and SF1126 in the MM.1R cell line. Optimal cell death was induced with bortezomib prior to SF1126, followed by concurrent administration. Immunoblot analysis of p-PDK1, downstream mTOR target (4E-BP1) were performed on the MM.1S cell line treated with 5, 10, 20, and 50uM SF1126 at 12 and 24 hours. At the 12 hour time point, p-PDK-1 appeared to increase, but was significantly reduced by 48 hours. A similar pattern of initial upregulation followed by reduction by 24 hours was seen with the mTOR protein 4E-BP1. Conclusion: SF1126 has dose dependent, in vitro activity in several multiple myeloma cell lines both as a single agent and in combination with dexamethasone, bortezomib, and melphalan. The addition of SF1126 to dexamethasone in a dexamethasone resistant cell line results in increased cell death, possibly by overcoming resistance mechanisms. The addition of SF1126 to bortezomib and melphalan also resulted in increased growth inhibition over either agent alone. These results warrant further study of this promising new pan-PI3K/Akt inhibitor.

L-asparaginase targets the protein translational mechanism and is an essential component in the treatment of acute lymphoblastic leukemia (ALL) and lymphoid malignancies. The current available preparations, E. coli and Erwinia have significant toxicities, often limiting their clinical use. In order to provide patients with a novel form of asparaginase, a recombinant form of Wolinella (rWS) asparaginase was developed. C3H mice were transplanted with 2 × 106 6C3HED lymphoma cells. Mice treated with native or rWS asparaginase demonstrated a significant decrease in tumor volume, and they remained tumor free for greater than 30 days after treatment. Human ALL cell lines were tested for sensitivity to rWS asparaginase and the molecular basis for rWS asparaginase sensitivity was examined in these cells. We hypothesized that rWS asparaginase would be immunologically distinct to that of E. coli. The sera of 6 patients previously identified to have high titer inhibitors to E. coli asparaginase were tested for antibodies to rWS asparaginase. The antibodies to E. coli did not bind to the rWS asparaginase, therefore demonstrating that there is no cross-reactivity to rWS asparaginase. Thus, antibodies or hypersensitivity reactions to previous forms of asparaginase will not prohibit therapy with rWS asparaginase. Therefore rWS asparaginase represents the first recombinant form of asparaginase and shows promise as a novel form of asparaginase which is immunologically distinct from E. coli and Erwinia asparaginase. In addition, it appears to be equally efficacious, and in previously published data, may have less toxicity. Asparaginase is an important component of treatment regimens for lymphoid malignancies, and therefore future studies will include a Phase I/II trial utilizing rWS asparaginase in relapsed lymphoid malignancies.

Background: Considerable evidence suggests an important role for the PI-3 kinase and AKT signaling pathways in survival and chemoresistance in multiple myeloma (MM) and other lymphoid malignancies. Our group and others have demonstrated that downregulation of p-AKT with combination therapy (bortezomib + lonafarnib; David et al, Blood, 2005) is a surrogate marker for myeloma apoptosis. It has been demonstrated that the compound, LY294002 has significant pan PI-3 kinase inhibitory properties but is not suitable for clinical use due to PK issues. SF1126 is a novel RGD targeted derivative of LY294002 that has been shown to have activity in a number of different tumor models. Herein, we evaluated the activity of SF1126 against the MM.1S and MM.1R MM cell lines in vitro and in vivo for sensitivity to PI-3 kinase inhibition. The results demonstrate that MM.1S and MM.1R tumor cell growth is sensitive to SF1126 with IC50 of 7.5 and 10.8 uM, respectively. The effects of SF1126 on MM.1R signaling in vitro was examined with profound inhibition of HIF1a induction under hypoxia, the suppression of phosphorylation states of MDM2, ERK and RS6 kinase. The IC50 for inhibition of p-AKT in MM.1S and MM.1R cells was determined to be 2.4 and 2.8 uM, respectively. SF1126 treatment (50 mg/kg/dose sc given every other day) inhibited MM.1R tumor growth in nude mouse xenografts 95% as compared to untreated controls on day 38 (p < .01). Microvessel density analysis of MM.1R tumor tissue demonstrated that SF1126 had significant antiangiogenic activity in vivo. Conclusion: The results provide preclinical data to support SF1126 as a clinically viable antiangiogenic, pan PI-3 kinase inhibitor for Phase I clinical trials in the treatment of multiple myeloma. Further studies in primary myeloma cells and in combination with conventional agents will be presented.