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Supplementary Figure 1. AZD5153 shows reduced BRD4 binding activity when BD2 function is abolished; Supplementary Figure 2. AZD5153 modulates MYC protein levels across hematologic cancer cell lines; Supplementary Figure 3. Gene Ontology (GO) analysis of the down-regulated transcripts by AZD5153 across all cell line types; Supplementary Figure 4. IC50 and percent maximal cell kill by AZD5153 and I-BET762 in five hematologic cell lines; Supplementary Figure 5. Tumor growth inhibition across six hematological xenograft Models

Protein modulation by AZD5153 in hematological cell lines

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The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563–74. ©2016 AACR.

Differentially expressed genes with AZD5153 treatment

Purpose: AZD5153 is a novel BRD4/BET inhibitor with a distinctive bivalent bromodomain binding mode. To support its clinical development, we identified pharmacodynamic (PD) biomarkers for use in clinical trials to establish target engagement.Experimental Design: CCR2 and CD180 mRNAs, initially identified from whole transcriptome profiling, were further evaluated by quantitative PCR in hematologic cell lines, xenografts, and whole blood from rat, healthy volunteers, and patients with cancer. MYC and HEXIM1 mRNAs were also evaluated.Results: RNA-sequencing data showed consistent decreases in CCR2/CD180 expression across multiple hematologic cell lines upon AZD5153 treatment. Evaluation of dose dependence in MV4,11 cells confirmed activity at clinically relevant concentrations. In vivo downregulation of CCR2/CD180 mRNAs (>80%) was demonstrated in MV4,11 and KMS-11 xenograft tumors at efficacious AZD5153 doses. Consistent with in vitro rat blood data, an in vivo rat study confirmed greater inhibition of CCR2/CD180 mRNA in whole blood versus MYC at an efficacious dose. Finally, in vitro treatment of whole blood from healthy volunteers and patients with cancer demonstrated, in contrast to MYC, almost complete downregulation of CCR2/CD180 at predicted clinically achievable concentrations.Conclusions: Our data strongly support the use of CCR2 and CD180 mRNAs as whole blood PD biomarkers for BRD4 inhibitors, especially in situations where paired tumor biopsies are unavailable. In addition, they can be used as tumor-based PD biomarkers for hematologic tumors. MYC mRNA is useful as a hematologic tumor-based biomarker but suboptimal as a whole blood biomarker. Utility of HEXIM1 mRNA may be limited to higher concentrations. Clin Cancer Res; 23(4); 1025–35. ©2017 AACR.

Figure S1: Downregulation of CCR2, CD180, MYC and upregulation of HEXIM1 mRNAs by AZD5153 in cells can be detected at 4h; Figure S2: Other BRD4/BET compounds can also downregulate CCR2 and CD180 mRNAs in hematological tumor cell lines; Supplementary Table 1: Description of cell lines used in the RNAseq study; Supplementary Table 2: Information on qPCR assays used in these studies; Supplementary Table 3: More detailed information for each healthy volunteer and patient sample.

Purpose:There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is critical to understand the potential for these molecules. However, it is challenging to measure tumor adenosine levels at scale, thus novel, clinically tractable biomarkers are needed.Experimental Design:We generated a gene expression signature for the adenosine signaling using regulatory networks derived from the literature and validated this in patients. We applied the signature to large cohorts of disease from The Cancer Genome Atlas (TCGA) and cohorts of immune checkpoint inhibitor–treated patients.Results:The signature captures baseline adenosine levels in vivo (r2 = 0.92, P = 0.018), is reduced after small-molecule inhibition of A2AR in mice (r2 = −0.62, P = 0.001) and humans (reduction in 5 of 7 patients, 70%), and is abrogated after A2AR knockout. Analysis of TCGA confirms a negative association between adenosine and overall survival (OS, HR = 0.6, P < 2.2e–16) as well as progression-free survival (PFS, HR = 0.77, P = 0.0000006). Further, adenosine signaling is associated with reduced OS (HR = 0.47, P < 2.2e–16) and PFS (HR = 0.65, P = 0.0000002) in CD8+ T-cell–infiltrated tumors. Mutation of TGFβ superfamily members is associated with enhanced adenosine signaling and worse OS (HR = 0.43, P < 2.2e–16). Finally, adenosine signaling is associated with reduced efficacy of anti-PD1 therapy in published cohorts (HR = 0.29, P = 0.00012).Conclusions:These data support the adenosine pathway as a mediator of a successful antitumor immune response, demonstrate the prognostic potential of the signature for immunotherapy, and inform patient selection strategies for adenosine pathway modulators currently in development.

2,4-Dinitroanisole (DNAN) is an important component of insensitive munitions that is anticipated to replace 2,4,6-trinitrotoluene (TNT) in munitions formulations. Photocatalyzed hydrogen peroxide (H2O2) oxidation experiments and chemical analyses were conducted to study the effect of initial pH and H2O2 dosage on the kinetics of DNAN decomposition and the reaction pathways. The results show that DNAN degradation followed zero-order kinetics when a 250 ppm DNAN solution was treated with ultraviolet (UV) light and 1500–4500 ppm H2O2 in an initial pH range of 4–7. However, when the H2O2 concentration was 750 ppm, DNAN degradation followed pseudo-first-order kinetics. The results indicate that DNAN can easily be oxidized by UV/H2O2 treatment. When the H2O2 dosage was 1500 ppm and the initial pH was 7, DNAN was reduced from 250 ppm to less than 1 ppm in 3 h. However, the total organic carbon (TOC) and total carbon (TC) concentrations were reduced slowly from 100 to less than 70 ppm carbon (C) in 3 h, and decreased to about 5 ppm after 9 h of treatment, suggesting the formation of other organic compounds. Those reaction intermediates were oxidized to carbon dioxide (CO2) at a slower rate than the oxidation of DNAN. CO2 was emitted from the solution because the solution pH decreased rapidly to about 3 during the UV/H2O2 oxidation. Most of the nitrogen in DNAN was converted to nitrate by UV/H2O2 oxidation after 9 h of treatment. The research results indicate that UV/H2O2 oxidation is a promising technique for the treatment of DNAN in wastewater. Keywords: 2,4-Dinitroanisole, Advanced oxidation processes, Wastewater treatment, Photocatalysis

Purpose: AZD5153 is a novel BRD4/BET inhibitor with a distinctive bivalent bromodomain binding mode. To support its clinical development, we identified pharmacodynamic (PD) biomarkers for use in clinical trials to establish target engagement.Experimental Design: CCR2 and CD180 mRNAs, initially identified from whole transcriptome profiling, were further evaluated by quantitative PCR in hematologic cell lines, xenografts, and whole blood from rat, healthy volunteers, and patients with cancer. MYC and HEXIM1 mRNAs were also evaluated.Results: RNA-sequencing data showed consistent decreases in CCR2/CD180 expression across multiple hematologic cell lines upon AZD5153 treatment. Evaluation of dose dependence in MV4,11 cells confirmed activity at clinically relevant concentrations. In vivo downregulation of CCR2/CD180 mRNAs (>80%) was demonstrated in MV4,11 and KMS-11 xenograft tumors at efficacious AZD5153 doses. Consistent with in vitro rat blood data, an in vivo rat study confirmed greater inhibition of CCR2/CD180 mRNA in whole blood versus MYC at an efficacious dose. Finally, in vitro treatment of whole blood from healthy volunteers and patients with cancer demonstrated, in contrast to MYC, almost complete downregulation of CCR2/CD180 at predicted clinically achievable concentrations.Conclusions: Our data strongly support the use of CCR2 and CD180 mRNAs as whole blood PD biomarkers for BRD4 inhibitors, especially in situations where paired tumor biopsies are unavailable. In addition, they can be used as tumor-based PD biomarkers for hematologic tumors. MYC mRNA is useful as a hematologic tumor-based biomarker but suboptimal as a whole blood biomarker. Utility of HEXIM1 mRNA may be limited to higher concentrations. Clin Cancer Res; 23(4); 1025–35. ©2017 AACR.

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563–74. ©2016 AACR.

CDK9 (cyclin-dependent kinase 9) regulates RNA transcription through its phosphorylation of RNA polymerase II and subsequent release from a "paused" elongation state, resulting in transcriptional activity. A transient inhibition of CDK9 activity will significantly impact transcripts with a short half-life, which includes the transcript for MCL1, an anti-apoptotic protein. AZD4573 is a highly potent and selective CDK9 inhibitor. We have previously reported that AZD4573 treatment results in dose-dependent inhibition of pSer2 of RNA polymerase II, reductions in MCL1 mRNA and protein, and an increase in cleaved caspase 3, resulting in cell death of hematological tumor cell lines and regressions in AML/DLBCL in vivo models [Cidado et. al., AACR Annual Meeting, Abstract 310 (2018)]. AZD4573 is currently being evaluated in a Phase I all-comers relapsed/refractory hematological malignancy trial (NCT03263637). To generate clinical evidence that the compound is inhibiting its target and having the expected downstream effects, we have developed pharmacodynamic biomarker (PD) assays for clinical use to measure the changes in pSer2/RNApol II, MCL1 protein, cleaved Caspase 3 and cleaved PARP. We have chosen a capillary electrophoresis platform which allows quantification and multiplexing while requiring lower sample quantities (80% inhibition of both pSer2 and MCL1 at 100nM (in 10% FCS). Next, we validated the use of these assays with human peripheral blood mononuclear cells (PBMCs) since we expected that some patients from the trial are likely to have significant numbers of circulating tumor cells (i.e. AML/CLL). After incubation of human whole blood with 1uM AZD4573 for different times, PBMCs were isolated and analyzed using the capillary electrophoresis assays. These results showed at least ~80% inhibition of pSer2 at 2h, consistent with preclinical efficacy modeling and predicted clinically achievable levels. In addition, we optimized the PBMC isolation protocol for the clinical sites and showed that compound washout during the procedure did not diminish AZD4573-mediated effects. In another study, we mimicked potential clinical conditions (e.g. 30 min, 1h, and 2h between blood collection and PBMC isolation, ambient vs 37°C storage conditions) and demonstrated that significant ex vivo apoptosis (i.e. activation of cleaved caspase 3) was unlikely to occur if PBMCs were isolated within 1-2h of blood collection. Finally, by showing that AZD4573 target engagement and pathway inhibition can be measured in normal PBMCs, these assays will be informative for all patients, including those without circulating tumor cells (e.g. many lymphomas). Here we describe the development and characterization of a suite of pharmacodynamic assays for clinical use with blood samples for our CDK9 program. Preliminary data from our Phase I study demonstrate successful implementation of these assays and support that AZD4573 inhibits CDK9 with the expected downstream effects, in the clinical setting. Updated results will be presented at the meeting. Disclosures Yeh: AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. Cidado:AstraZeneca: Employment, Equity Ownership. Barrett:AstraZeneca: Employment, Equity Ownership.

Unions and policing are a relatively under‐researched area. But this paper, adopting a comparative approach, argues that police unions have been and still are influential, that they differ across cultures, and are involved in a variety of ways in police reform. They can be supportive of change, hostile to it, or closely involved in the reform process. Some are representative bodies where the police may not unionize while there are increasingly special interest groups based on faith, gender, and ethnicity. This paper focuses on representation and voice for ordinary officers in relation to reform in policing.

Summary Molecular genetics in lower organisms has allowed the elucidation of pathways that modulate the aging process. In certain instances, evolutionarily conserved genes and pathways have been shown to regulate lifespan in mammals as well. Many gene products known to affect lifespan are intimately involved in the control of energy metabolism, including the fuel sensor AMP-activated protein kinase (AMPK). We have shown previously that over-expression of an AMPK α subunit in Caenorhabditis elegans, designated aak-2, increases lifespan. Here we show the interaction of aak-2 with other pathways known to control aging in worms. Lifespan extension caused by daf-2/insulin-like signaling mutations was highly dependent on aak-2, as was the lifespan extension caused by over-expression of the deacetylase, sir-2.1. Similarly, there was partial requirement for aak-2 in lifespan extension by mitochondrial mutations (isp-1 and clk-1). Conversely, aak-2 was not required for lifespan extension in mutants lacking germline stem cells (glp-1) or mutants of the eating response (eat-2). These results show that aging is controlled by overlapping but distinct pathways and that AMPK/aak-2 represents a node in a network of evolutionarily conserved biochemical pathways that control aging.

Although limiting energy availability extends lifespan in many organisms, it is not understood how lifespan is coupled to energy levels. We find that the AMP:ATP ratio, a measure of energy levels, increases with age in Caenorhabditis elegans and can be used to predict life expectancy. The C. elegans AMP-activated protein kinase α subunit AAK-2 is activated by AMP and functions to extend lifespan. In addition, either an environmental stressor that increases the AMP:ATP ratio or mutations that lower insulin-like signaling extend lifespan in an aak-2-dependent manner. Thus, AAK-2 is a sensor that couples lifespan to information about energy levels and insulin-like signals.

Metal-on-metal hip resurfacing was developed for younger, active patients as an alternative to THA, but it remains controversial. Study heterogeneity, inconsistent outcome definitions, and unstandardized outcome measures challenge our ability to compare arthroplasty outcomes studies.We asked how early revisions or reoperations (within 5 years of surgery) and overall revisions, adverse events, and postoperative component malalignment compare among studies of metal-on-metal hip resurfacing with THA among patients with hip osteoarthritis. Secondarily, we compared the revision frequency identified in the systematic review with revisions reported in four major joint replacement registries.We conducted a systematic review of English language studies published after 1996. Adverse events of interest included rates of early failure, time to revision, revision, reoperation, dislocation, infection/sepsis, femoral neck fracture, mortality, and postoperative component alignment. Revision rates were compared with those from four national joint replacement registries. Results were reported as adverse event rates per 1000 person-years stratified by device market status (in use and discontinued). Comparisons between event rates of metal-on-metal hip resurfacing and THA are made using a quasilikelihood generalized linear model. We identified 7421 abstracts, screened and reviewed 384 full-text articles, and included 236. The most common study designs were prospective cohort studies (46.6%; n = 110) and retrospective studies (36%; n = 85). Few randomized controlled trials were included (7.2%; n = 17).The average time to revision was 3.0 years for metal-on-metal hip resurfacing (95% CI, 2.95-3.1) versus 7.8 for THA (95% CI, 7.2-8.3). For all devices, revisions and reoperations were more frequent with metal-on-metal hip resurfacing than THA based on point estimates and CIs: 10.7 (95% CI, 10.1-11.3) versus 7.1 (95% CI, 6.7-7.6; p = 0.068), and 7.9 (95% CI, 5.4-11.3) versus 1.8 (95% CI, 1.3-2.2; p = 0.084) per 1000 person-years, respectively. This difference was consistent with three of four national joint replacement registries, but overall national joint replacement registries revision rates were lower than those reported in the literature. Dislocations were more frequent with THA than metal-on-metal hip resurfacing: 4.4 (95% CI, 4.2-4.6) versus 0.9 (95% CI, 0.6-1.2; p = 0.008) per 1000 person-years, respectively. Adverse event rates change when discontinued devices were included.Revisions and reoperations are more frequent and occur earlier with metal-on-metal hip resurfacing, except when discontinued devices are removed from the analyses. Results from the literature may be misleading without consistent definitions, standardized outcome metrics, and accounting for device market status. This is important when clinicians are assessing and communicating patient risk and when selecting which device is most appropriate for individual patients.

Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins and is frequently amplified or over-expressed in both solid tumors and hematological malignancies, suggesting that its activity may be important for the survival of cancer cells. CDK9 inhibition results in the down regulation of Mcl-1 mRNA and subsequent protein levels by inhibiting transcription and represents an indirect approach to targeting Mcl-1. Mcl-1 can also be targeted directly using an inhibitor that disrupts the Mcl-1 complexes to induce apoptosis. Using both molecular and pharmacological approaches, we sought to identify predictive biomarkers of Mcl1 dependency in sensitive NSCLC and multiple myeloma cell lines. Here we demonstrate that NSCLC cell lines lacking MCL1 gene copy number gains are not sensitive to siRNA mediated knockdown of Mcl-1 or Mcl-1 inhibition (cell line sensitivity to CDK9 or Mcl-1 inhibition is defined by potency and extent of caspase activation). However, the presence of a copy number alteration does not predict sensitivity to Mcl-1 inhibition. To better understand what the drivers of sensitivity are, we developed quantitative assays on the Peggy platform (a capillary based immunoassay platform by Protein Simple) to measure both Mcl-1 and Bcl-xL protein levels. Using these assays, we show a correlation between sensitivity to a CDK9 or Mcl1 inhibitor and Mcl-1 levels, as well as to the ratio of Mcl-1 to Bcl-xL protein in a NSCLC cell line panel. These findings were then extended into a panel of multiple myeloma cell lines. While somewhat broad activity for CDK9 or Mcl-1 inhibition is seen across the cell lines tested, a subset of the sensitive lines have MCL1 amplification and express high levels of Mcl-1 protein. Mcl-1 levels alone, however, do not predict for sensitivity across the panel and, similar to NSCLC, the ratio of Mcl1 to Bcl-xL expression has greater positive predictive value. These results provide the rationale for exploring Mcl-1 copy number alterations and Mcl-1 and Bcl-xL protein levels as predictive biomarkers for tumor response when treating with a CDK9 or Mcl-1 inhibitor in both NSCLC and multiple myeloma. Citation Format: Kristen McEachern, Greg O’Connor, Justin Cidado, Matthew Belmonte, Evan Barry, Hannah Dry, Paul Secrist, Lisa Drew. Predicting response to Mcl-1 targeting agents in NSCLC and multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3558.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is the primary constituent in the explosive munitions that are produced at Holston Army Ammunition Plant (HSAAP), Kingsport, TN. Part of a modernization effort for the 66-year-old plant includes the goal of zero RDX discharge to the facility industrial wastewater treatment plant. This study examines multiple technologies for removing RDX from the process waste stream at Holston Army Ammunition Plant. The treatment technologies evaluated included granular activated carbon (GAC), anoxic biotreatment, zerovalent iron (ZVI), alkaline hydrolysis, ultra-violet oxidation, and electrochemical decomposition. Evaluation criteria include capital and operating cost, and effectiveness in transforming RDX to nontoxic end products. Based on laboratory assessments using site water, alkaline hydrolysis and electrochemical treatment were selected for pilot scale evaluation. The RDX removal half-lives for the selected technologies were on the order of 0.25 hr with decomposition of RDX from 10,000 micrograms/L to less than 20 micrograms/L.

Dry and wet physical separation processes were tested at Yuma Proving Ground to remove depleted uranium (DU) from soil. Four sample locations were tested that had varied uranium concentration, weathering, and aging of fired, DU residues. Reduction of soil DU concentration was achieved using simple vibratory or agitated screening techniques. For soils into which the DU had been recently fired, these techniques were successful at removing a large fraction (>70 percent) of the total uranium present (by mass). A heavy liquid separation process based on a water/sodium polytungstate solution was tested. This produced a sinking fraction that contained nearly 100-percent uranium and uranium oxide by mass for the less weathered soils. However, this type of wet separation is not currently practical for field use. A water-based separation process using an angled vibrating table to facilitate gravity transport of separated DU was also tested. This method produced a fraction of concentrated uranium along with fractions of soil particles with reduced densities. However, this process required extensive particle size separation prior to use and produced a contaminated waste stream that required secondary treatment. The extent to which DU and DU residues could be removed from the Yuma soils depended on the extent of soil weathering and corrosion of the DU alloy.

Infection after total hip replacement (THR) adversely affects patients' function and health-related quality of life (HRQL). A prosthesis with antibiotic-loaded acrylic cement (PROSTALAC) was designed to improve the function and quality of life of patients undergoing treatment for infected THR.We assessed 23 patients with the PROSTALAC implant in situ for treatment of an infected THR for function and HRQL, using standardized outcome measures. These patients were compared with a referent cohort of patients who had undergone assessment of function and HRQL before and 6 months after primary THR in the same tertiary health centres.The mean (standard deviation) Western Ontario MacMaster (WOMAC) scores for PROSTALAC patients were 70.0 (21.1), 65.8 (20.4) and 63.0 (21.1) for pain, stiffness and function, respectively. The median Harris Hip score was 62.3 (minimum 20.4, maximum 86.3) and median global hip range of motion was 100.0 (minimum 80.0, maximum 140.0) degrees.The mean WOMAC scores for pain, stiffness and function were better than they were for patients awaiting THR but not as good as 6 months after primary THR. The PROSTALAC implant allows patients to have reasonable function and quality of life during the interim treatment for deep joint infection.

Pim kinases have been shown to play a key role as downstream effectors of growth factor signaling in hematological malignancies. We have previously described AZD1208, a novel, orally bioavailable, highly selective pan Pim kinase inhibitor, undergoing clinical testing in AML. Specifically, we had demonstrated the anti-proliferative activity of AZD1208 in models of AML and had shown that AZD1208 treatment of AML cell lines results in the dose dependent reduction of pBAD at serine 112 and p4EBP1 at serine 65 as well as pp70S6K at threonine 389 and pS6 at serine 235/236, associated with global effects on protein translation (Keeton et al. Blood 2014). Since AZD1208 is about 10-15 fold more potent against Pim-1 than Pim-2 in both enzymatic and cellular assays, we sought to assess the dependency of each of these markers on Pim-1 and/or Pim-2 for phosphorylation using a Pim-1/3 selective inhibitor (Pim-1/3) that lacks Pim-2 inhibitory activity. In vitro studies demonstrate that while the modulation of pBAD does not require inhibition of Pim-2, the regulation of 4EBP1 as well as p70S6K and S6 are dependent, at least in part, on Pim-2 activity. As these proteins are canonically regulated by mTORC1, these data are consistent with Pim-2 acting upstream of this complex; perhaps through phosphorylation of TSC2 as reported for multiple myeloma cell lines (Lu et al. Blood 2013). These observations are also supported by in vivo data. Analysis of pBAD and p4EBP1 levels in Molm16 xenografts show that maximal inhibition of pBAD (70-80% inhibition) is achieved at AZD1208 plasma concentration of about 1000 ng/ml, consistent with the estimated cellular IC90 of pBAD inhibition in vitro. However, maximal inhibition of p4EBP1 is achieved at concentrations 3-5 fold higher. This observation is consistent with the differential potency of AZD1208 against Pim-1 and Pim-2, and with a requirement for Pim-2 inhibition for maximal p4EBP1 inhibition in this AML model. pBAD and p4EBP1 were selected as the pharmacodynamic endpoints for evaluating clinical target inhibition in patient samples. Bone marrow and peripheral blood samples were collected pre- and post-dose from relapsed, refractory AML patients enrolled in the AZD1208 Phase I dose escalation. Analysis of pBAD inhibition on day 1 of dosing shows at least 60-70% inhibition across all of the dosing cohorts. With clinical exposures on day 1 approaching 1000 ng/ml at the first dose level and exceeding 1000 ng/ml as the dose increases, the extent of pBAD inhibition seen in patients appears to be consistent with Pim-1 inhibition, as seen in preclinical models. Furthermore, and also similar to our preclinical observations, maximal inhibition of p4EBP1 in patients is achieved only at higher exposures. These data strengthen the hypothesis that BAD phosphorylation is primarily dependent on Pim-1, whereas suppression of Pim-2 activity is required for maximal inhibition of p4EBP1 in AML cells. In summary, Pim inhibition in AML cell line models and in patients treated with AZD1208 results in the inhibition of the downstream targets of Pim signaling, pBAD and p4EBP1. Invitro and in vivo, the inhibition of pBAD is consistent with inhibition of Pim-1 while inhibition of p4EBP1 indicates a requirement for Pim-2 inhibition as well. These observations are validated in patients and provide further evidence for the relevance of these biomarkers as a measure of Pim signaling in AML. Disclosures McEachern: AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. DuPont:AstraZeneca: Employment, Equity Ownership. Gibbons:AstraZeneca: Employment, Equity Ownership. Pablo:AstraZeneca: Employment, Equity Ownership. Vishwanathan:AstraZeneca: Employment, Equity Ownership. McCoon:AstraZeneca: Employment, Equity Ownership. Cortes:AstraZeneca: Research Funding. Neumann:AstraZeneca: Employment, Equity Ownership. Keating:AstraZeneca: Employment, Equity Ownership. Pease:AstraZeneca: Employment, Equity Ownership. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Barrett:AstraZeneca: Employment, Equity Ownership.

Abstract 3720 Spleen tyrosine kinase (SYK) plays a key role in B cell receptor mediated survival in certain B cell malignancies including Diffuse Large B Cell Lymphoma (DLBCL). Therefore, targeting SYK represents an emerging therapeutic approach for the treatment of DLBCL. Indeed, fostamatinib, an orally available SYK inhibitor, has shown promising clinical activity in non-Hodgkin lymphoma (Friedberg et al. 2010 Blood 115: 2578). However, the overall response rate for novel targeted agents in unselected relapsed refractory DLBCL patients remains 25–30%, highlighting the opportunity for development of better treatment strategies. Here, we used preclinical models to study the mechanism underlining the efficacy of R406, the metabolic active form of fostamatinib. R406 was characterized in a heterogeneous panel of 17 DLBCL cell lines including both ABC and GCB subtypes. Overall, the cellular selectivity of R406, particularly in ABC-subtype DLBCL cell lines, was consistent with other BCR-targeted agents such as BTK inhibitor PCI-32765 (ibrutinib) and PI3 kinase δ inhibitor CAL-101. This strongly suggests that R406 functions through inhibition of BCR-mediated survival signaling. Furthermore, phospho-flow analysis and Western Blotting have demonstrated the effect of R406 on both basal and anti-IgG/M stimulated BCR signaling. Specifically, R406 decreases phosphorylation of proximal BCR pathway regulators BLNK, PLCγ2, as well as key players in downstream effect pathways such as ERK, AKT, RPS6, 4EBP and STAT3. To investigate whether the inhibition of BCR signaling by R406 affects cell viability, flow-cytometry based apoptosis analysis was employed. R406 induced apoptosis in sensitive cell lines in a dose-dependent manner. Mechanistically, treatment with R406 reduced MCL1 protein level and down-regulated Bfl-1 expression in sensitive, but not resistant, cell lines, which may partially contribute to the observed efficacy. Given the critical role of NFκB-dependent survival signaling in ABC subtype DLBCL, we then investigated the effect of R406 on this pathway by monitoring NF-κB target gene expression via quantitative real time PCR. Consistent with previous publication (Davis et al. 2010, Nature 463: 88), R406 significantly blocked chronic active BCR-induced NFκB signaling in sensitive cell lines with CD79A/B mutations, leading to downregulation of IL-6/IL-10 and subsequent suppression of JAK/STAT3 signaling. In contrast, we observed little effect of R406 in cell lines with downstream activating mutations of the NFκB pathway (such as CARD11, A20 mutations). Therefore, the molecular nature of NF-κB pathway lesions may serve as a predictive marker for R406 responsiveness in ABC subtype DLBCL cell lines. Furthermore, in exploration of a common indicator of sensitivity to R406 in both ABC and GCB subtypes, we have confirmed that response to R406 is dependent on surface expression of a functional BCR and presence of a intact BCR signaling cascade (Chen et al. 2008 Blood 111: 2230). These cells are thus characterized as “B-cell Receptor active” or BCR subtype. Work is currently underway to further elucidate the characteristics of the BCR subtype, which may serve as a general selective marker for R406's efficacy in DLBCL cell lines. In conclusion, we have demonstrated that R406 functions as a BCR antagonist and reduces viability in DLBCL through inhibition of NFκB-mediated survival signals and downregulation of MCL-1. In addition, we have confirmed in ABC subtype DLBCL cell lines, similar to other BCR-targeted agents, R406 sensitivity is correlated with CD79A/B mutations whereas A20 or CARD11 mutations render cells resistance. Eventually, we proposed that BCR classification may serve as a broader selection maker for all DLBCL cell lines. Disclosures: Zhang: AstraZeneca: Employment, Equity Ownership. Parmentier:AstraZeneca: Employment, Equity Ownership. Lai:AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. Passino:AstraZeneca: Employment, Equity Ownership. Powell:AstraZeneca: Employment, Equity Ownership. Devereaux:AstraZeneca: Employment, Equity Ownership. Byth:AstraZeneca: Employment, Possible shareholder Other.