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TRAIL can activate cell surface death receptors, resulting in potent tumor cell death via induction of the extrinsic apoptosis pathway. Eftozanermin alfa (ABBV-621) is a second generation TRAIL receptor agonist engineered as an IgG1-Fc mutant backbone linked to two sets of trimeric native single-chain TRAIL receptor binding domain monomers. This hexavalent agonistic fusion protein binds to the death-inducing DR4 and DR5 receptors with nanomolar affinity to drive on-target biological activity with enhanced caspase-8 aggregation and death-inducing signaling complex formation independent of FcγR-mediated cross-linking, and without clinical signs or pathologic evidence of toxicity in nonrodent species. ABBV-621 induced cell death in approximately 36% (45/126) of solid cancer cell lines in vitro at subnanomolar concentrations. An in vivo patient-derived xenograft (PDX) screen of ABBV-621 activity across 15 different tumor indications resulted in an overall response (OR) of 29% (47/162). Although DR4 (TNFSFR10A) and/or DR5 (TNFSFR10B) expression levels did not predict the level of response to ABBV-621 activity in vivo, KRAS mutations were associated with elevated TNFSFR10A and TNFSFR10B and were enriched in ABBV-621–responsive colorectal carcinoma PDX models. To build upon the OR of ABBV-621 monotherapy in colorectal cancer (45%; 10/22) and pancreatic cancer (35%; 7/20), we subsequently demonstrated that inherent resistance to ABBV-621 treatment could be overcome in combination with chemotherapeutics or with selective inhibitors of BCL-XL. In summary, these data provide a preclinical rationale for the ongoing phase 1 clinical trial (NCT03082209) evaluating the activity of ABBV-621 in patients with cancer. Significance: This study describes the activity of a hexavalent TRAIL-receptor agonistic fusion protein in preclinical models of solid tumors that mechanistically distinguishes this molecular entity from other TRAIL-based therapeutics.

Flow cytometry is a powerful technique for the detection and quantification of cell surface and intracellular proteins. It enables the ability to measure the expression levels of specific proteins in a cell population of interest without the need to physically separate out the cells from within a heterogeneous population by using the appropriate cell-specific markers. It also requires fewer cells than other traditional techniques such as Western blotting. Here we describe a robust and reproducible method to measure the expression levels of the BCL-2 family members, BCL-2, BCL-XL, and MCL-1 by quantitative flow cytometry (QFCM) using validated antibodies.

Background We have developed a quantitative fluorescence cytometry (QFCM) method that can be used to measure BCL-2 family member proteins in cell lines and clinical samples. We described the validation of antibodies, methods development and application of the assay. Method We characterized and validated antibodies to BCL-2, BCL-XL, and MCL-1 in cell lines to confirm specificity for flow cytometry. Each protein was measured in a panel of leukemia/lymphoma cell lines and B-cells from chronic lymphocytic leukemia (CLL) patients treated with the BCL-2/BCL-XL inhibitor navitoclax. The cellular activity of various BCL-2 family member inhibitors alone and in combination was determined to demonstrate utility of our assay to correlate protein levels with efficacy. Results We identified antibodies that were highly specific for each protein. The expression profile in cell lines as determined by molecules of equivalent soluble fluorochrome was comparable to western blot. Using our assay, BCL-2, BCL-XL, and MCL-1 protein levels were shown to correlate with response to BCL-2 family inhibitors in vitro and could be measured in clinical samples. Conclusions This method can quantify BCL-2 family members in a specific, highly reproducible and sensitive fashion, and requires fewer cells compared to western blot. It is particularly useful for identifying BCL-2, BCL-XL, and MCL-1 protein levels in a specific cell population within a heterogeneous population like those collected from CLL patients. These data show that our QFCM method can be used to facilitate the quantification and evaluation of biomarkers predictive of response in patients treated with BCL-2 family member inhibitors. © 2016 International Clinical Cytometry Society

Cell death can be initiated through activation of the extrinsic and intrinsic apoptotic signaling pathways. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, preferentially triggers the extrinsic apoptotic pathway by binding as a trimer to two closely related cell surface death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Receptor trimerization leads to the formation of the death-inducing signaling complex (DISC) to recruit and activate downstream caspases that ultimately leads to apoptotic cell death. Because TRAIL signaling induces apoptosis, several TRAIL receptor agonists have been developed for the treatment of cancer. ABBV-621 is a novel, second generation TRAIL receptor agonist that is an engineered fusion protein consisting of an IgG1-Fc linked to a single chain trimer of TRAIL subunits resulting in a total of six death receptor binding sites per molecule to maximize receptor clustering that is currently being tested in Phase I clinical trials (NCT03082209). To expand upon the potential therapeutic utility of ABBV-621, we tested the combinatorial activity of ABBV-621 with numerous standard-of-care (SoC) therapeutics and targeted agents in diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Thein vitroresults led to selection of agents to combine with ABBV-621 forin vivostudies. In DLBCL cell line-derived xenograft (CDX) preclinical models, we observed combination activity of ABBV-621 with pevonedistat (PEV) a selective NEDD8 inhibitor. Additionally, synergistic activity was observed with ABBV-621 with either bendamustine (BED) or rituximab (RTX) alone, or BED/RTX together. In AML, we observed compelling combination activity of ABBV-621 with PEV in cell line-derived xenograft (CDX) models. In MM, combination of ABBV-621 plus bortezomib (BTZ) resulted in deeper anti-tumorigenic activity than either agent alone in several CDX models. The pre-clinical data presented here support expanding the indications and settings where ABBV-621 may have utility. A clinical trial assessing the activity of ABBV-621 in combination with bortezomib and dexamethasone in R/R MM patients is planned. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Disclosures Smith: AbbVie:Current Employment, Current equity holder in publicly-traded company.Jin:AbbVie:Current Employment, Current equity holder in publicly-traded company.Chen:AbbVie:Current Employment, Current equity holder in publicly-traded company.Zhang:AbbVie:Current Employment, Current equity holder in publicly-traded company.Huska:AbbVie:Current Employment, Current equity holder in publicly-traded company.Widomski:AbbVie:Current Employment, Current equity holder in publicly-traded company.Bontcheva:AbbVie:Current Employment, Current equity holder in publicly-traded company.Buchanan:AbbVie:Current Employment, Current equity holder in publicly-traded company.Morgan-Lappe:AbbVie:Current Employment, Current equity holder in publicly-traded company.Phillips:AbbVie:Current Employment, Current equity holder in publicly-traded company.Tahir:AbbVie:Current Employment, Current equity holder in publicly-traded company.

Background Venetoclax (ABT-199), a first-in-class orally bioavailable BCL-2-selective inhibitor, was recently approved by the FDA for use in patients with 17p-deleted chronic lymphocytic leukemia who have received prior therapy. It is also being evaluated in numerous clinical trials for treating patients with various hematologic malignancies. As with any targeted cancer therapy, it is critically important to identify potential mechanisms of resistance, both for patient stratification and developing strategies to overcome resistance, either before it develops or as it emerges. Methods In order to gain a more comprehensive insight into the nature of venetoclax resistance mechanisms, we evaluated the changes in the BCL-2 family members at the genetic and expression levels in seven different venetoclax-resistant derived leukemia and lymphoma cell lines. Results Gene and protein expression analyses identified a number of different alterations in the expression of pro- and anti-apoptotic BCL-2 family members. In the resistant derived cells, an increase in either or both the anti-apoptotic proteins BCL-XL or MCL-1, which are not targeted by venetoclax was observed, and either concomitant or exclusive with a decrease in one or more pro-apoptotic proteins. In addition, mutational analysis also revealed a mutation in the BH3 binding groove (F104L) that could potentially interfere with venetoclax-binding. Not all changes may be causally related to venetoclax resistance and may only be an epiphenomenon. For resistant cell lines showing elevations in BCL-XL or MCL-1, strong synergistic cell killing was observed when venetoclax was combined with either BCL-XL- or MCL-1-selective inhibitors, respectively. This highlights the importance of BCL-XL- and MCL-1 as causally contributing to venetoclax resistance. Conclusions Overall our study identified numerous changes in multiple resistant lines; the changes were neither mutually exclusive nor universal across the cell lines tested, thus exemplifying the complexity and heterogeneity of potential resistance mechanisms. Identifying and evaluating their contribution has important implications for both patient selection and the rational development of strategies to overcome resistance. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3383-5) contains supplementary material, which is available to authorized users.

A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.

Sirolimus (rapamycin) is an immunosuppressant used in preventing allograft rejection and in drug-eluting stents to prevent restenosis after angioplasty. Zotarolimus, an analogue of sirolimus, was designed to have a shorter in vivo half-life. Zotarolimus was found to be mechanistically similar to sirolimus in having high-affinity binding to the immunophilin FKBP12 and comparable potency for inhibiting in vitro proliferation of both human and rat T cells. Rat pharmacokinetic studies with intravenous dosing demonstrated terminal elimination half-lives of 9.4 hours and 14.0 hours for zotarolimus and sirolimus, respectively. Given orally, T1/2 values were 7.9 hours and 33.4 hours, respectively. Consistent with its shorter duration, zotarolimus showed a corresponding and statistically significant 4-fold reduction in potency for systemic immunosuppression in 3 rat disease models. Pharmacokinetic studies in cynomolgus monkey underpredicted the half-life difference between zotarolimus and sirolimus apparent from recent clinical data. In vitro inhibition of human coronary artery smooth muscle cell proliferation by zotarolimus was comparable to sirolimus. Drug-eluting stents for local delivery of zotarolimus to the vessel wall of coronary arteries are in clinical development. The pharmacological profile of zotarolimus suggests it may be advantageous for preventing restenosis with a reduced potential for causing systemic immunosuppression or other side effects.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.

The Bcl-2 family inhibitors venetoclax and navitoclax demonstrated potent antitumor activity in chronic lymphocytic leukemia patients, notably in reducing marrow load and adenopathy. Subsequent trials with venetoclax have been initiated in non-Hodgkin's lymphoma and multiple myeloma patients. Traditional preclinical models fall short either in faithfully recapitulating disease progression within such compartments or in allowing the direct longitudinal analysis of systemic disease. We show that intravenous inoculation of engineered RS4;11 (acute lymphoblastic leukemia) and Granta 519 (mantle cell lymphoma) bioluminescent reporter cell lines result in tumor engraftment of bone marrow, with additional invasion of the central nervous system in the case of Granta 519. Importantly, apoptosis induction and response of these systemically engrafted tumors to Bcl-2 family inhibitors alone or in combination with standard-of-care agents could be monitored longitudinally with optical imaging, and was more accurately reflective of the observed clinical response.

Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein we report a series of MCL-1 inhibitors that emanated from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compounds from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compound 30b rapidly induced caspase activation with associated loss in cell viability. The small molecules described herein thus comprise effective tools for studying MCL-1 biology.

NFAT (nuclear factor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca(2+)/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN activity thus inhibiting nuclear translocation of NFAT and consequent cytokine gene transcription. The inhibition of CaN in cells outside of the immune system may contribute to the toxicities associated with cyclosporin A therapy. In a search for safer immunosuppressive drugs, we identified a series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2 cytokine gene transcription. The BTP compounds block the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT confirmed that the BTP compounds block calcium-induced movement of NFAT from the cytosol to the nucleus. Inhibition of NFAT was selective because the BTP compounds did not affect the activation of NF-kappaB and AP-1 transcription factors. Treatment of intact T cells with the BTP compounds prior to calcium ionophore-induced activation of CaN caused NFAT to remain in a highly phosphorylated state. However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT by CaN in vitro, nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor Elk-1. The data suggest that the BTP compounds cause NFAT to be maintained in the cytosol in a phosphorylated state and block the nuclear import of NFAT and, hence, NFAT-dependent cytokine gene transcription by a mechanism other than direct inhibition of CaN phosphatase activity. The novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection.

Several 1,2,3,4-tetrahydro- and 7-N-hydroxycarbamate derivatives of the natural product rapamycin were prepared and assayed for their immunosuppressive and antifungal profiles. Substitutions at the 7-position indicate the possibility of a differentiated immunosuppressive to antifungal profile, whereas 40-position variants of the tetrahydro-analogues did not show similar differentiated activity.

T lymphocytes play a critical part in inflammatory skin diseases but are targeted by available therapies that have only partial efficacy, significant side-effects, or both. Because psoriasis, atopic dermatitis, and allergic contact hypersensitivity are associated with T helper type 1 (Th1), T helper type 2 (Th2), or mixed Th1–Th2 cell subsets and cytokine types, respectively, there is a need for a better broad-based inhibitor. The macrolactam ascomycin analog, ABT-281, was found to inhibit potently T cell function across species and to inhibit expression of multiple cytokines in human peripheral blood leukocytes which have been found in human skin disease cells and tissues. These included immunoregulatory Th1 (interleukin-2 and interferon-γ) and Th2 (interleukin-4 and interleukin-5) cytokines. ABT-281 was shown to have potent topical activity (ED 50 = 0.6% in acetone/olive oil) in a stringent swine model of allergic contact hypersensitivity, but its potency was markedly reduced compared with ascomycin when administered systemically due to more rapid clearance. Topical application of 3% ABT-281 in acetone/olive oil over 25% of the body surface in swine resulted in undetectable blood levels. Compared with a wide potency range of topical corticosteroids in clinical formulations, 0.3% and 1% ABT-281 ointments profoundly inhibited dinitrochlorobenzene-induced contact hypersensitivity in the pig by 78% and 90%, respectively, whereas super-potent steroids such as clobetasol propionate only inhibited in the 50% range and mild to moderate potency steroids such as fluocinolone acetonide were inactive. The potent topical activity of ABT-281 in swine, its superior efficacy, its rapid systemic clearance following uptake into the bloodstream, and its ability to inhibit cytokine biosynthesis of both Th1 and Th2 cell subsets, suggests that it will have a broad therapeutic value in inflammatory skin diseases, including psoriasis, atopic dermatitis, and allergic contact dermatitis.

To assess the duration of immunosuppression in FK506-dosed pigs, an undiluted whole blood assay was established to measure reactivities of T cells in their physiological milieu. PMA and ionomycin were shown to induce IL-2 production in swine blood. The IC50of FK506 in inhibiting IL-2 production in whole blood and isolated PBMC stimulated with PMA and ionomycin measured 1.2 and 0.04 nM, respectively. These data underscore the influence of red blood cells and plasma proteins on drug potency. IL-2 levels were determined in blood drawn immediately before and 1, 24, 48, and 72 h after iv dosing. For pigs dosed with 0.05 mg/kg, 50% recovery of IL-2 production was observed at 16 h and 100% at 35 h after dosing. For pigs dosed with 0.15 mg/kg, 50% recovery was observed at 38 h and 100% at 72 h. Blood concentrations of FK506 at 50 and 100% recovery of IL-2 production measured 10.8 and 2.2 nM for pigs dosed with 0.05 mg/kg and 6.1 and 1.1 nM for pigs dosed with 0.15 mg/kg, respectively. These concentrations are severalfold higher than predicted from the IC50of FK506 for inhibiting IL-2 production in the whole blood assay. These data suggest that the true potency of FK506 in blood after dosing is influenced by additional factors, which could include plasma protein binding, the presence of active or interfering metabolites, serum interfering factors, and sequestration of drug in blood cells. Our results demonstrate the utility of an undiluted whole blood assay for assessing the duration of immunosuppression in drug-dosed animals and emphasize the importance of assessing drug potency in the whole blood environmentex vivo.

This study examined psychosocial correlates of immune function and illness in 89 male first-year US Air Force Academy cadets. A psychosocial questionnaire was administered to cadets prior to their arrival at the academy and was readministered during cadet orientation and during the stressful environment of Basic Cadet Training (BCT). Immune responsiveness was analyzed by PHA-, PMA-, or anti-CD3-stimulated thymidine uptake in mononuclear leucocytes. Illness episodes were assessed via medical chart review and self-reported symptoms. There were significant increases in distress levels as cadets entered BCT. No psychosocial measure assessed prior to arrival at the academy predicted level of PHA-, PMA-, and anti-CD3-stimulated thymidine uptake or risk of illness. However, hostility levels reported during BCT predicted risk of illness in the four weeks following psychosocial assessment (odds ratio = 7.1; 95% confidence interval: 1.4-36.1). Elevated response to environmental stressors and lower well-being levels also predicted impending illness, but only in the cohort of cadets who had not contracted food poisoning prior to assessment during BCT (OR = 9.3, CI = 1.9-46.7; OR = 0.09, CI = 0.02-0.53). These results suggest that self-report measures of hostility, response to environmental stressors and well-being may be useful predictors of impending illness episodes in males encountering high stress environments.

The ability of a cancer cell to avoid apoptosis is crucial to tumorigenesis and can also contribute to chemoresistance. The Bcl-2 family of prosurvival proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1) plays a key role in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small-molecule inhibitor of Bcl-2, Bcl-XL, and Bcl-w. While navitoclax exhibits single-agent activity in tumors dependent on Bcl-2 or Bcl-XL for survival, the expression of Mcl-1 has been shown to confer resistance to navitoclax, most notably in solid tumors. Thus, therapeutic agents that can downregulate or neutralize Mcl-1 are predicted to synergize potently with navitoclax. Here, we report the activity of navitoclax in combination with 19 clinically relevant agents across a panel of 46 human solid tumor cell lines. Navitoclax broadly enhanced the activity of multiple therapeutic agents in vitro and enhanced efficacy of both docetaxel and erlotinib in xenograft models. The ability of navitoclax to synergize with docetaxel or erlotinib corresponded to an altered sensitivity of the mitochondria toward navitoclax, which was associated with the downmodulation of Mcl-1 and/or upregulation of Bim. These data provide a rationale to interrogate these combinations clinically. Mol Cancer Ther; 10(12); 2340–9. ©2011 AACR.

ABT-737 and ABT-263 are potent inhibitors of the BH3 antiapoptotic proteins, Bcl-x(L) and Bcl-2. This class of putative anticancer agents invariantly contains an acylsulfonamide core. We have designed and synthesized a series of novel quinazoline-based inhibitors of Bcl-2 and Bcl-x(L) that contain a heterocyclic alternative to the acylsulfonamide. These compounds exhibit submicromolar, mechanism-based activity in human small-cell lung carcinoma cell lines in the presence of 10% human serum. This comprises the first successful demonstration of a quinazoline sulfonamide core serving as an effective benzoylsulfonamide bioisostere. Additionally, these novel quinazolines comprise only the second known class of Bcl-2 family protein inhibitors to induce mechanism-based cell death.

Revision rhinoplasty can present difficult and challenging problems, which maximally test the skill and judgment of the facial plastic surgeon. We conducted a retrospective study of 1221 consecutive rhinoplasties, of which 170 were revision procedures. The rate of revision of our own procedures is 5.3%. Postrhinoplastic deformities are divided anatomically into the upper, middle, and lower thirds of the nose. The largest category of deformities occur in the lower thirds of the nose, in which bossa are the most common problem. Overall, pollybeak is the most common deformity in 33% of the procedures performed, followed by bossa in 26%, and excessive dorsal removal in 24%. We analyzed the causes and selected management of these cases.

ABT-199 is a first-in-class orally bioavailable BCL-2 selective inhibitor that is currently being testing in clinical trials. Clinical activity has been reported in chronic lymphocytic leukemia and NHL patients treated with ABT-199 as a monotherapy. As with any targeted cancer therapy, it is important to identify the potential mechanisms of resistance, not only to inform patient selection but also to develop strategies to circumvent resistance as it emerges. In this study, we generated resistant variants from ABT-199-sensitive cell lines of different leukemia and lymphoma subtypes (two diffuse large B-cell lymphomas, two follicular lymphomas, one leukemia line, and two mantle cell lymphomas) by increasing exposure to ABT-199 in a stepwise manner over time. Compared to the parental cell lines, the variants were 10 to >100-fold less sensitive to ABT-199. The ABT-199-resistant variants were also resistant to ABT-263 (navitoclax), a BCL-2/BCL-XL inhibitor, as well as to other standard-of-care cancer agents. Gene and protein expression analyses revealed multiple alterations in the expression of BCL-2 family members in the resistant variants; however, not all of the changes were universal. Alterations included changes in the levels of the target protein BCL-2, reduction of the pro-apoptotic proteins BAX, BIM or NOXA, or increases in the anti-apoptotic proteins BCL-XL or MCL-1. In some but not all cases, the changes to gene and protein expression levels were accompanied by changes in DNA copy number. To determine whether changes in the anti-apoptotic BCL-2 family members were causally related to ABT-199 resistance, we tested ABT-199 in combination with BCL-XL and MCL-1 selective inhibitors. Co-treating with ABT-199 and either a BCL-XL or MCL-1 selective compound was synergistic in killing the ABT-199-resistant variants, suggesting these proteins can both contribute directly to ABT-199 resistance. Under appropriate selection pressure, intrinsic traits that mitigate the effects of anticancer drugs can lead to the enrichment of a resistant tumor population. In ABT-199-resistant RS4;11 cells we observed a complete and permanent loss of BAX, one of the ultimate effectors of mitochondrial mediated apoptosis. FACS analysis revealed that there was a pre-existing population of RS4;11 cells lacking this protein. It is likely that these cells had a selective advantage over those expressing BAX when treated with ABT-199. Overall our data indicate that there are distinct mechanisms of resistance to ABT-199, either innate or acquired, which will have important implications for both patient selection and developing strategies to overcome resistance. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for the research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Stephen K. Tahir, Morey L. Smith, Paul Hessler, Lisa Roberts-Rapp, Joel D. Leverson, Lloyd T. Lam. Mechanisms of resistance to ABT-199 in leukemia and lymphoma cell lines. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B30.

All authors are employees of AbbVie and participated in the design, conduct, and interpretation of these studies. AbbVie and Genentech provided financial support for these studies and participated in the review and approval of this publication. The BCL-2-selective inhibitor ABT-199 has demonstrated efficacy in numerous preclinical models of hematologic malignancies without causing thrombocytopenia, a dose-limiting toxicity associated with the BCL-2/BCL-XL inhibitor navitoclax (Souers et al. 2013. Nat. Med. 19, 202-208). ABT-199 has also demonstrated clinical activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) (Seymour et al. 2014. J. Clin. Oncol. 32, 448s; Davids et al. 2014. J. Clin. Oncol. 32, 544s). Despite these encouraging early clinical data, some subjects do not respond to ABT-199 or progress while on treatment. Pre-clinical models indicate that both intrinsic and acquired resistance may be a consequence of MCL-1 expression. Consequently, we have explored potent and selective small molecule inhibitors of CDK9, a kinase known to maintain the expression of MCL-1 through its role in p-TEFb-mediated transcription. Inhibition of CDK9 resulted in the rapid loss in RNA polymerase II phosphorylation (Serine 5) and MCL-1 expression that was closely followed by the induction of apoptosis in MCL-1-dependent cell lines, a cellular response that could be rescued by overexpression of BCL-2. Substantial synergy was observed between CDK9 inhibitors and ABT-199 in a number of hematologic cell lines with intrinsic or acquired resistance to ABT-199. Direct inhibition of MCL-1 with the small molecule BH3 mimetic A-1210477 was also highly synergistic with ABT-199, further validating the utility of co-inhibiting MCL-1 and BCL-2 function simultaneously in ABT-199 resistant tumors. Importantly, the CDK9 inhibitor-ABT-199 combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in xenograft models of non-Hodgkin’s lymphoma (NHL) and acute myelogenous leukemia (AML). These data indicate that CDK9 inhibitors may be highly efficacious when used in combination with ABT-199 for the treatment of hematologic malignancies. Disclosures Chen: Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Tapang:abbvie: Employment, Equity Ownership. Tahir:abbvie: Employment, Equity Ownership. Smith:abbvie: Employment, Equity Ownership. Xue:abbvie: Employment, Equity Ownership. Zhang:abbvie: Employment, Equity Ownership. Gao:abbvie: Employment, Equity Ownership. Tong:abbvie: Employment, Equity Ownership. Clark:abbvie: Employment, Equity Ownership. Ricker:abbvie: Employment, Equity Ownership. Penning:abbvie: Employment, Equity Ownership. Albert:abbvie: Employment, Equity Ownership. Phillips:abbvie: Employment, Equity Ownership. Souers:abbvie: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership.

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-γ were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-γ secretion by Hut 78 cells or CD3+ T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.

In a search for novel early T cell activation transcripts, we identified expressed sequence tags (ESTs) more abundantly expressed in normal human CD4+ T lymphocytes fully activated by a 5 h exposure to CD3 plus CD28 mAbs, compared to the same cells stimulated with either CD3 mAb or CD28 mAb alone. An EST was identified that hybridized with a 1.7 kb transcript expressed in activated T cells but was undetectable by Northern blot analysis in resting T cells or other normal tissues. The T cell transcript was maximally induced within 6 h and remained elevated for at least 47 h. Induction of the transcript was blocked by cyclosporin A, FK506, and dexamethasone but not by rapamycin. The transcript was polyadenylated but lacked an open reading. A BLAST search of the NCBI database revealed that the transcript shared identity with the recently reported human BIC proto-oncogene that encodes a noncoding mRNA (W. Tam, Gene 274 (2001) 157). Our data demonstrate that transcriptional activation of the BIC proto-oncogene is an early and sustained T cell activation event and suggest an important role for noncoding mRNA in T cell function.

Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.

Multiple environmental factors asociated with space flight can increase the risk of infectious illness among crewmembers thereby adversely affecting crew health and mission success. Host defences can be impaired by multiple physiological and psychological stressors including: sleep deprivation, disrupted circadian rhythms, separation from family, perceived danger, radiation exposure, and possibly also by the direct and indirect effects of microgravity. Relevant human immunological data from isolated or stressful environments including spaceflight will be reviewed. Long-duration missions should include reliable hardware which supports sophisticated immunodiagnostic capabilities. Future advances in immunology and molecular biology will continue to provide therapeutic agents and biologic response modifiers which should effectively and selectively restore immune function which has been depressed by exposure to environmental stressors.

We recently disclosed the discovery of ABT-737, a potent antagonist of antiapoptotic Bcl-2 family proteins that induced tumor regression in murine xenograft models. ABT-263 is a related, orally bioavailable Bcl-2 family protein inhibitor that is currently in clinical development. For both agents, multiple daily dosing from two to four weeks was well tolerated in all species evaluated. The primary preclinical toxicological finding in mouse, rat and dog was a unique thrombocytopenia characterized by a rapid clearance of circulating platelets. A series of in vivo studies that were performed to better characterize this phenomenon are described here. After a single intravenous or oral dose in dogs, circulating platelet counts decreased rapidly and concentration dependently with a nadir at approximately six hours post administration. Platelet counts returned to near normal levels within several days post dose, accompanied by an increase in both mean platelet volume and percent of reticulated platelets. Analysis of platelets by aggregometry during this rebound period indicated that returning platelets were fully functional. Following multiple doses, platelet counts also decreased rapidly after initial dosing, but exhibited evidence of rebound during the dosing period that appeared to be dose dependant. Evaluation of fibrinogen, d-dimer, antithrombin-III, PT and APTT did not support a mechanism involving consumptive coagulopathy. Whole body scintography utilizing [111] Indium labeled platelets in dogs indicated that platelet clearance after compound administration is primarily mediated by the liver. These data suggest that ABT-737 and ABT-263 preferentially affect circulating platelets rather than abrogating platelet production in the bone marrow. In fact, the observed rebound in platelet count in the face of continued dosing reflects the capacity for the animals to compensate for the effect. The enantiomer of ABT-737 (which has significantly lower activity against Bcl-2 family proteins) had no effect on circulating platelets in animals suggesting an underlying mechanism that is dependent on inhibition of antiapoptotic Bcl-2 family proteins. The unusually rapid kinetics of platelet clearance and recovery suggests that the drug-induced thrombocytopenia elicited by these Bcl-2 family protein inhibitors is unique compared to that observed with conventional cytotoxic chemotherapy.

Comparing the bones that make up each side of the nose by anthropometry reveals asymmetries caused by both developmental and traumatic processes, which precludes performing osteotomies using a set procedure. The frontal process of the maxilla is a key structure in narrowing the bony contours of the nose to correct the “open roof syndrome” (incomplete closure of the roof of the nose) after hump removal. It must be included in many instances in order to achieve a satisfactory infracture. This is especially important in the management of a large or asymmetric frontal process. Furthermore, osteotomies should be placed sufficiently lateral to the nasal bones to prevent the aesthetically unpleasing complication of the so-called step deformity. The questionable efficacy of osteotomies in rhinoplasty on the non-Caucasian nose is also briefly considered in this article.

Rhinoplasty is one of the most challenging surgical procedures. Surgeons should strive to obtain maximum aesthetic and functional results with minimal operative intervention. Excessive surgery often results in unwarranted impairment of nasal function. The obstructive or sensory complications of rhinoplasty can occur in the early or late postoperative periods. The most common functional sequelae of rhinoplasty, and their causes, prevention and therapy, are discussed.

A modification of an absorbent, non-adherent material (Telfa) is described for use as an anterior nasal packing. The ideal nasal packing should fulfill certain criteria. It should be easy to introduce and remove, contour to the nasal cavity to exert a tamponade effect, and should not prolapse or react unfavorably with the mucous membranes of the nose. The advantages of Telfa as a nasal packing is discussed and compared to previously described materials in this regard. During the past three years this pack has been used in over 800 patients for anterior epistaxis, septal and rhinoplastic surgery. The results have been extremely satisfactory, and the authors suggest their use in these cases.

A method of chin augmentation employing a newer synthetic material (Proplast®)‡ is presented. The characteristics and biocompatibility of Proplast are detailed. Technical considerations and results of short term clinical trials in 20 patients are discussed. One implant necessitated removal secondary to trauma four months after surgery. All other implants including over 30 additional cases have been without incident.

Often in rhinoplasty, adequate osteotomies and mobilization of the nasal bones fail to close the dorsum--leaving an open roof deformity. Preexisting conditions and certain anatomical relationships contribute to this problem. The various methods of correcting the deformity are discussed and the author's approach presented.

• Double lateral osteotomy has proved to be useful in the management of broad, thick, and heavy maxillary processes. The technique of performing two lateral osteotomies and the indications for its application are described. The results obtained from cadaver dissection and clinical experience with this technique are discussed. ( Arch Otolaryngol 103:344-348, 1977)

The functional sequelae of rhinoplastic surgery are emphasized and the functional and anatomical causes of postoperative nasal obstruction are discussed. A maneuver to improve the airway and decrease the incidence of postoperative nasal obstruction is presented.

The difficulties in obtaining an adequate result in the Negroid nose have been elucidated by other authors. Paucity of lobular cartilage; the flat dorsum; short columella; wide flaring nares; and skin that tends to keloid formation have led many surgeons to attempt radical surgical techniques to obtain rather limited results. To circumvent the complications in the more radical procedures, a more conservative concept of an entirely intranasal operation without external skin incisions is outlined. This technique consists of tip rotation and lobular cartilage trimming; the use of a non-absorbable basal bunching suture, and the placement of a dorsal implant without performing osteotomies. A comparison with other techniques is made, as well as surgical aims, limitations and a discussion of results. Pertinent illustrations and photographs are presented.

The concept of rhinoplasty of the mature nose is presented. As an isolated procedure or in conjunction with other facial plastic procedures, shortening the length of a drooping nose can give the effect of a more youthful appearance. The anatomy and physiology of the nose as it ages is discussed and if understood by the surgeon, these patients need no longer be rejected because of their age. Technical consideration are important, and the use of the modified rim incision, high septal transfixion and lobule rotation is suggested. An analysis of 30 cases over a noe year period is documented to support this thesis.

• Many materials have been used for augmentation mentoplasty with varying degrees of success. In our experience with the silicone bag-gel implant in 50 cases over the past 18 months the results have been quite satisfactory and there were no untoward complications. ( Arch Otolaryngol 103:292-293, 1977)

Satisfactory treatment of alopecia following rhytidectomy is discussed using the punch graft technique. In spite of the presence of scar tissue following rhytidectomy, this procedure has been quite successful because of the rich blood supply in that area.

Different types of alloplastic implants are currently being utilized in performing mentoplasty. A review of the literature points out the number of prostheses that have been used. Each type of material has inherit physical properties which determine its characteristics for use as a chin implant. The type of implant and method selected in chin augmentation depends upon accurate preoperative evaluation and full understanding of the properties of alloplastic substances. Five hundred and thirty-nine cases of chin augmentation utilizing different materials and methods were reviewed over a nine year period. The limitations of mentoplasty as well as the advantages, disadvantages, and in selected cases, the indications for the use of a particular implant are discussed.

The advantages of the Fomon technique modifications in rhinoplasty are outlined. The importance of these adaptations is emphasized.

Recognition of plasma cell tumors of the upper respiratory system and particularly of the accessory nasal sinuses has increased since attention has been called to their incidence by New, 1 Claiborn, 2 Jackson 3 and others. The otolaryngologist finds these tumors of particular interest because of their occurrence in the upper respiratory tract and, especially, in the maxillary sinuses. The plasma cell tumor is generally considered to arise from the bone marrow although many cases of extraosseous origin have been reported. The classic multiple myeloma is thought to be a disease of the bone marrow or of the blood-forming structures generally, and, while the plasma cell tumors seem to be a phase in the developmental history of multiple myeloma, the cause and nature of these tumors are not definitely known and their exact relationship to multiple myeloma 4 is not clear. If the plasma cell tumor should be regarded as