Antagonist ABT-737 Research Articles

The super elongation complex drives transcriptional addiction in MYCN-amplified neuroblastoma.

MYCN amplification in neuroblastoma leads to aberrant expression of MYCN oncoprotein, which binds active genes promoting transcriptional amplification. Yet, how MYCN coordinates transcription elongation to meet productive transcriptional amplification and which elongation machinery represents MYCN-driven vulnerability remain to be identified. We conducted a targeted screen of transcription elongation factors and identified the super elongation complex (SEC) as a unique vulnerability in MYCN-amplified neuroblastomas. MYCN directly binds EAF1 and recruits SEC to enhance processive transcription elongation. Depletion of EAF1 or AFF1/AFF4, another core subunit of SEC, leads to a global reduction in transcription elongation and elicits selective apoptosis of MYCN-amplified neuroblastoma cells. A combination screen reveals SEC inhibition synergistically potentiates the therapeutic efficacies of FDA-approved BCL-2 antagonist ABT-199, in part due to suppression of MCL1 expression, both in MYCN-amplified neuroblastoma cells and in patient-derived xenografts. These findings identify disruption of the MYCN-SEC regulatory axis as a promising therapeutic strategy in neuroblastoma.

Synergistic Interactions between the Hypomethylating Agent Thio-Deoxycytidine and Venetoclax in Myelodysplastic Syndrome Cells

Interactions between the novel hypomethylating agent (HMA) thio-deoxycytidine (T-dCyd) and the BCL-2 antagonist ABT-199 (venetoclax) have been examined in human myelodysplastic syndrome (MDS) cells. The cells were exposed to agents alone or in combination, after which apoptosis was assessed, and a Western blot analysis was performed. Co-administration of T-dCyd and ABT-199 was associated with the down-regulation of DNA methyltransferase 1 (DNMT1) and synergistic interactions documented by a Median Dose Effect analysis in multiple MDS-derived lines (e.g., MOLM-13, SKM-1, and F-36P). Inducible BCL-2 knock-down significantly increased T-dCyd's lethality in MOLM-13 cells. Similar interactions were observed in the primary MDS cells, but not in the normal cord blood CD34+ cells. Enhanced killing by the T-dCyd/ABT-199 regimen was associated with increased reactive oxygen species (ROS) generation and the down-regulation of the anti-oxidant proteins Nrf2 and HO-1, as well as BCL-2. Moreover, ROS scavengers (e.g., NAC) reduced lethality. Collectively, these data suggest that combining T-dCyd with ABT-199 kills MDS cells through an ROS-dependent mechanism, and we argue that this strategy warrants consideration in MDS therapy.

Artificial intelligence-based identification of octenidine as a Bcl-xL inhibitor

Apoptosis plays an essential role in maintaining cellular homeostasis and preventing cancer progression. Bcl-xL, an anti-apoptotic protein, is an important modulator of the mitochondrial apoptosis pathway and is a promising target for anticancer therapy. In this study, we identified octenidine as a novel Bcl-xL inhibitor through structural feature-based deep learning and molecular docking from a library of approved drugs. The NMR experiments demonstrated that octenidine binds to the Bcl-2 homology 3 (BH3) domain-binding hydrophobic region that consists of the BH1, BH2, and BH3 domains in Bcl-xL. A structural model of the Bcl-xL/octenidine complex revealed that octenidine binds to Bcl-xL in a similar manner to that of the well-known Bcl-2 family protein antagonist ABT-737. Using the NanoBiT protein–protein interaction system, we confirmed that the interaction between Bcl-xL and Bak-BH3 domains within cells was inhibited by octenidine. Furthermore, octenidine inhibited the proliferation of MCF-7 breast and H1299 lung cancer cells by promoting apoptosis. Taken together, our results shed light on a novel mechanism in which octenidine directly targets anti-apoptotic Bcl-xL to trigger mitochondrial apoptosis in cancer cells.

The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function

Maintaining the architecture, size and composition of an intact stem cell (SC) compartment is crucial for tissue homeostasis and regeneration throughout life. In mammalian skin, elevated expression of the anti-apoptotic Bcl-2 protein has been reported in hair follicle (HF) bulge SCs (BSCs), but its impact on SC function is unknown. Here, we show that systemic exposure of mice to the Bcl-2 antagonist ABT-199/venetoclax leads to the selective loss of suprabasal BSCs (sbBSCs), thereby disrupting cyclic HF regeneration. RNAseq analysis shows that the pro-apoptotic BH3-only proteins BIM and Bmf are upregulated in sbBSCs, explaining their addiction to Bcl-2 and the marked susceptibility to Bcl-2 antagonism. In line with these observations, conditional knockout of Bcl-2 in mouse epidermis elevates apoptosis in BSCs. In contrast, ectopic Bcl-2 expression blocks apoptosis during HF regression, resulting in the accumulation of quiescent SCs and delaying HF growth in mice. Strikingly, Bcl-2-induced changes in size and composition of the HF bulge accelerate tumour formation. Our study identifies a niche-instructive mechanism of Bcl-2-regulated apoptosis response that is required for SC homeostasis and tissue regeneration, and may suppress carcinogenesis.

BCL-2 Antagonist ABT-199 Combined with Complex I Inhibitor IACS-010759 Blocks Mitochondrial Respiration and Facilitates Anti-Leukemia Efficacy in Pre-Clinical AML Models

ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that activates pro-apoptotic BCL-2 family proteins causing cell death. BCL-2 family regulates ADP/ATP exchange across the mitochondrial membrane by voltage-dependent anion channel (VDAC) to prevent the loss of coupled mitochondrial respiration during the apoptosis (Manfredi, J Bio Chem 2003). We and other have shown that AML is BCL-2 dependent and leukemia stem cell (LSC) rely on BCL-2 to regulate the oxidative phosphorylation (OxPhos) activity for survival (Pan, Cancer Discovery 2014; Lagadinou, Cell Stem Cell, 2013). IACS-010759, a novel complex I inhibitor, effectively inhibits cell respiration and exhibits potent anti-leukemia effect in AML (Molina, 2016 AACR abstract #335) . We hypothesized that BCL-2 facilitates OxPhos activity, and that BCL-2 inhibition with ABT-199 will enhance anti-leukemia efficacy of IACS-010759 in AML.Priming of AML cell lines with ABT-199 (0-33 nM) for 24 hrs, followed by IACS-010759 (0-33 nM) for 72 hrs drastically inhibited viable cell numbers as determined by flow cytometry. Analysis of cellular respiration using Seahorse showed that both ABT-199 at 20nM and IACS-010759 at 11nM partially (<15%) reduced oxygen consumption rate (OCR), while combination treatment fully (60%) inhibited basal and maximal OCR as early as 2 hrs. This translated into inhibition of ATP production at 6 hrs and loss of the mitochondrial membrane potential (MMP) measured by TMRE/MTG or JC-1 flow cytometry. Further, ABT-199 followed by IACS-010759 induced cleavage of caspase-3 and DNA damage shown by g-H2AX immunoblotting. Anti-BCL-2 immunoprecipitation showed decreased BCL-2/VDAC binding in ABT-199- and IACS-010759-treated cells (Fig 1A). Sequential treatment with ABT-199 followed by IACS-010759 (10-100 nM) in primary AML cells (n=3) caused additive decrease in viable cell numbers.To explore the role of BCL-2 in mitochondrial metabolism, we utilized HL-60 cells overexpressing BCL-2. The HL-60/BCL-2 cells (a kind gift by Dr. Kapil N. Bhalla) have increased OxPhos activity as measured by basal and maximal OCR by Seahorse (Fig.1B); and increased basal and mitochondrial ROS production measured by H2DCFDA and MitoSOX Red flow cytometry. BCL-2 inhibition by ABT-199 at 100 nM after 2 hrs treatment induced ROS production in control but not in HL-60/BCL-2 cells. Further, HL-60/BCL-2 cells were less sensitive to IACS-010759 compared to control cells. These data suggest that BCL-2 facilities OxPhos maintenance and reduces efficacy of mitochondrial inhibitors.We next tested the combined anti-AML efficacy of BCL-2 and OxPhos inhibitors in an in vivo leukemia model, by injecting NSG mice with AML PDX cells harboring FLT3-ITD. Upon engraftment, leukemia-bearing mice were randomized to receive vehicle; single agent ABT-199 at 100 mg/kg for 14 days; IACS-010759 at 7.5 mg/kg for 21 days; or sequential treatment with ABT-199 for 14 days followed by IACS-010759 for 21 days. Consistent with in vitro findings, mice in the IACS-010759 and IACS-010759/ABT-199 combination groups had lower circulating tumor burden compared to other groups as measured by hCD45% flow cytometry (Fig. 1C). Survival analysis in all groups is ongoing. Tumor cells collected from 2 mice of ABT-199, IACS-010759, or the combination treatment group at the end of drug dosing showed decreased OCR. The frequency of CD34+CD38-CD123+ AML stem/progenitor cells was decreased in ABT-199, IACS-010759 and combination groups, with notable induction of differentiation in combination group (CD11b and CD11c), measured by CyTOF mass cytometry (Fig. 1D).In summary, our data indicate that in addition to its known anti-apoptotic function, BCL-2 facilitates mitochondrial respiration and OxPhos in AML cells, possibly through direct binding of mitochondrial pore protein VDAC. Inhibition of BCL-2 with ABT-199 disrupts BCL-2/VDAC interactions, reduces OCR and induces ROS production. Combined blockade of BCL-2 with ABT-199 and of mitochondrial complex I with IACS-010759 produces a greater anti-AML efficacy in AML cells in vitro and in vivo . [Display omitted] DisclosuresBattula:United Therapeutics Corporation: Research Funding.

The Pan-FLT3/BTK Multi-Kinase Inhibitor CG '806 Induces AML Killing in FLT-Mutant and Wild Type Cells, and Exerts Synergistic Pro-Apoptotic Effects with Concomitant Targeting of Anti-Apoptotic Bcl-2 and/or Mcl-1

Internal tandem duplication (ITD) or point mutations of the Fms-like tyrosine kinase 3 (FLT3) are present in approximately 30% of patients with acute myeloid leukemia (AML) (Thiede et al., 2002). The ITD mutation is associated with very poor prognosis (Kottaridis et al., 2003) and additional acquired mutations of FLT3, including the D835 or the “gatekeeper” F691 mutation, have been identified in patients who developed resistance to currently used FLT3 inhibitors (Man et al., 2012; Smith et al., 2012). In addition, the upregulation of FLT3 ligand (FL) resulting from conventional chemotherapy, or bone marrow (BM) microenvironmental-mediated protection of AML cells, are also associated with resistance to FLT3-targeted therapy (Sato et al., 2011).CG'806 is a first-in-class inhibitor that demonstrates sub-nanomolar activity against FLT3, Bruton's tyrosine kinase (BTK), aurora kinases, and certain other kinase family clusters in cell-free systems. We evaluated the anti-leukemia effect of CG'806 in human and murine leukemia cell lines harboring FLT3-ITD mutations and determined induced cell growth inhibition and apoptosis at sub-nanomolar to low nanomolar levels. Importantly, CG'806 also exerted pronounced anti-leukemia activity against cells with D835 TKD and FLT3-ITD plus F691L, Y842D or D835 dual mutations. CG'806 triggered marked apoptosis in vitro primary AML patient samples with and without FLT3-ITD mutations. Mechanistically, CG'806 demonstrated profound suppression of phospho-FLT3, -BTK and -aurora kinases, and their downstream signaling pathways (Zhang et al., AACR Hematological Malignancies, 2017). In addition, CG'806 completely abrogated protection mediated by FL or BM stromal cells in FLT3 ITD mutated MOLM14 cells, while quizartinib and sorafenib activity was markedly reduced in co-culture with mesenchymal stromal cells (MSCs). In a Baf3-ITD cell-engrafted murine leukemia model, CG'806 (delivered orally Qdx5 each week using a primitive formulation) significantly reduced leukemia burden, eliminated leukemic blasts, and extended survival in a dose-dependent manner versus the (vehicle) control.Interestingly, CG'806 did not modulate anti-apoptotic proteins Bcl-2 levels, but upregulated anti-apoptotic Mcl-1, albeit marked cleavage of caspase-3 and PARP was observed in murine Baf3-FLT3-ITD mutated cells. Hence, we tested combinations of CG'806 plus the Bcl-2 antagonist ABT199, the Mcl-1 inhibitor A1210477, or both. All combinations showed marked synergistic pro-apoptotic effects in both FLT3 wild type (WT) and mutated leukemic cells, including those cells harboring dual or “gatekeeper” mutations (e.g., CIs were 0.41 ± 0.04, 0.26 ± 0.04, 0.59 ± 0.09 and 0.90 ± 0.02 in FLT3 WT, ITD, ITD+F691L and ITD+D835Y cells, respectively, in triple combo of CG'806, ABT199 and A1210477).In summary, CG'806 was effective in both FLT3 WT and mutant AML cells, including those harboring dual ITD plus D835/F691 mutations. CG'806 also abrogated AML cell protection mediated by FL or BM stromal cells. Concomitant blockade of FLT3 and Bcl-2 or Mcl-1 synergistically induced pro-apoptotic effects in both FLT3 WT and mutant leukemic cells including dual mutant cells. CG'806 is under development for the treatment of patients with FLT3 WT and FLT3-mutated AML. [Display omitted] DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Glycine ameliorates mitochondrial dysfunction caused by ABT-199 in porcine oocytes.

Mitochondria play an important role in controlling oocyte developmental competence. Our previous studies showed that glycine (Gly) can regulate mitochondrial function and improve oocyte maturation in vitro. However, the mechanisms by which Gly affects mitochondrial function during oocyte maturation in vitro have not been fully investigated. In this study, we induced a mitochondrial damage model in oocytes with the Bcl-2-specific antagonist ABT-199. We investigated whether Gly could reverse the mitochondrial dysfunction caused by ABT-199 exposure and whether it is related to calcium regulation. Our results showed that ABT-199 inhibited cumulus expansion, decreased the oocyte maturation rate and the intracellular glutathione (GSH) level, caused mitochondrial dysfunction, which was confirmed by decreased mitochondrial membrane potential (ΔΨm) and the expression of mitochondrial function-related genes PGC-1α, and increased reactiveoxygenspecies (ROS) levelsand the expression of apoptosis-associated genes Bax, Caspase-3, and Cyto C.More importantly, ABT-199-treated oocytes showed an increase in the intracellular free calcium concentration ([Ca2+]i) and had impaired cortical type 1 inositol 1,4,5-trisphosphate receptors (IP3R1) distribution. Nevertheless, treatment with Gly significantly ameliorated mitochondrial dysfunction, oxidative stress, and apoptosis, and Gly also regulated [Ca2+]i levels and IP3R1 cellular distribution, which further protects oocyte maturation in ABT-199-induced porcine oocytes.Taken together, our results indicate that Gly has a protective action against ABT-199-induced mitochondrial dysfunction in porcine oocytes.

Low‑dose trametinib and Bcl‑xL antagonist have a specific antitumor effect in KRAS‑mutated colorectal cancer cells.

KRAS‑mutant colorectal cancer (CRC) is a highly malignant cancer with a poor prognosis, however specific therapies targeting KRAS mutations do not yet exist. Anti‑epidermal growth factor receptor (EGFR) agents, including cetuximab and panitumumab, are effective for the treatment of certain patients with CRC. However, these anti‑EGFR treatments have no effect on KRAS‑mutant CRC. Therefore, new therapeutic strategies targeting KRAS‑mutant CRC are urgently needed. To clarify the direct effect of KRAS gene mutations, the present study transduced mutant forms of the KRAS gene (G12D, G12V and G13D) into CACO‑2 cells. A drug‑screening system (Mix Culture assay) was then applied, revealing that the cells were most sensitive to the MEK inhibitor trametinib among tested drugs, Cetuximab, Panitumumab, Regorafenib, Vemurafenib, BEZ‑235 and Palbociclib. Trametinib suppressed phosphorylated ERK (p‑ERK) expression and inhibited the proliferation of KRAS‑mutant CACO‑2 cells. However, low‑dose treatment with trametinib also increased the expression of the anti‑apoptotic protein Bcl‑xL in a dose‑dependent manner, leading to drug resistance. To overcome the resistance of KRAS‑mutant CRC to apoptosis, the combination of trametinib and the Bcl‑xL antagonist ABT263 was assessed by invitro and invivo experiments. Compared with the effects of low‑dose trametinib monotherapy, combination treatment with ABT263 had a synergistic effect on apoptosis in mutant KRAS transductants invitro. Furthermore, invivo combination therapy using low‑dose trametinib and ABT263 against a KRAS‑mutant (G12V) xenograft synergistically suppressed growth, with an increase in apoptosis compared with the effects of trametinib monotherapy. These data suggest that a low dose of trametinib (10nM), rather than the usual dose of 100nM, in combination with ABT263 can overcome the resistance to apoptosis induced by Bcl‑xL expression, which occurs concurrently with p‑ERK suppression in KRAS‑mutant cells. This strategy may represent a promising new approach for treating KRAS‑mutant CRC.

BCL-2 antagonism sensitizes cytotoxic T cell-resistant HIV reservoirs to elimination ex vivo.

Curing HIV infection will require the elimination of a reservoir of infected CD4+ T cells that persists despite HIV-specific cytotoxic T cell (CTL) responses. Although viral latency is a critical factor in this persistence, recent evidence also suggests a role for intrinsic resistance of reservoir-harboring cells to CTL killing. This resistance may have contributed to negative outcomes of clinical trials, where pharmacologic latency reversal has thus far failed to drive reductions in HIV reservoirs. Through transcriptional profiling, we herein identified overexpression of the prosurvival factor B cell lymphoma 2 (BCL-2) as a distinguishing feature of CD4+ T cells that survived CTL killing. We show that the inducible HIV reservoir was disproportionately present in BCL-2hi subsets in ex vivo CD4+ T cells. Treatment with the BCL-2 antagonist ABT-199 was not sufficient to drive reductions in ex vivo viral reservoirs when tested either alone or with a latency-reversing agent (LRA). However, the triple combination of strong LRAs, HIV-specific T cells, and a BCL-2 antagonist uniquely enabled the depletion of ex vivo viral reservoirs. Our results provide rationale for novel therapeutic approaches targeting HIV cure and, more generally, suggest consideration of BCL-2 antagonism as a means of enhancing CTL immunotherapy in other settings, such as cancer.

A-1210477, a selective MCL-1 inhibitor, overcomes ABT-737 resistance in AML.

Acute myeloid leukemia (AML) is one of the most common hematological malignancies. It is difficult to treat since it easily develops resistance to therapeutic drugs. Myeloid cell leukemia 1 (MCL-1), BCL-2 and BCL-XL, which belong to the anti-apoptotic group of proteins in the BCL-2 family, are overexpressed in AML. The effects of inhibitors that target anti-apoptotic proteins of the BCL-2 family in AML were evaluated in the present study. MCL-1 protein levels of HL60, MOLM13, OCI-AML3 and MV4-11 cell lines were investigated. Furthermore, following treatment with MCL-1-selective antagonist A-1210477 and/or BCL-2/BCL-XL antagonist ABT-737, cell viability was detected. The chimera rate of human CD45(+) cells of bone marrow from mouse models was analyzed via flow cytometry and immunohistochemistry using murine tissues (lung, spleen and liver). The data revealed that the HL-60 cell line, which exhibited a low MCL-1 protein level, and MOLM-13 and MV4-11 cell lines, whose MCL level was intermediate, were sensitive to ABT-737, whereas OCI-AML3 cells, which exhibited a high MCL-1 level, were insensitive to ABT-737. However, multiple AML mouse models and AML cell lines were sensitive to the MCL-1-selective antagonist A-1210477. The results of the present study indicated that the MCL-1-selective antagonist could overcome the resistance to the BCL-2/BCL-XL antagonist (ABT-737) in vitro and in vivo.

Abstract 1854: Novel pateamine analogs to target the translation initiation factor eIF4A in chronic lymphocytic leukemia

Abstract The viability of chronic lymphocytic leukemia (CLL) is critically dependent upon staving off death by apoptosis, a hallmark of CLL pathophysiology. The overexpression of the Bcl-2 family proteins likely play a major role in the apoptosis blockade in CLL, and has been an effective target of CLL therapy. The recognition that Mcl-1, a major component of the anti-apoptotic response, is intrinsically short-lived and must be continually resynthesized suggested a novel therapeutic approach. Pateamine A (PatA), a macrolide marine natural product, inhibits cap-dependent translation by binding to the initiation factor eIF4A. We have previously reported the first total synthesis of PatA. Mechanistic studies suggested that binding of eIF4A to PatA caused the stalling of initiation complexes on mRNA, halting the translation initiation process. In this study, we demonstrated that a synthetic derivative of PatA, des-methyl des-amino PatA (DMDAPatA), blocked mRNA translation, reduced Mcl-1 protein and initiated apoptosis in CLL cells. This action was synergistic with the Bcl-2 antagonist ABT-199, by a mechanism to inhibit the two parallel arms of apoptosis control in CLL. However, avid binding to human plasma proteins limited DMDAPatA potency, precluding further development. To address this, we synthesized a new series of 27 PatA analogs with modifications on various regions of PatA, and screened their toxicity against the primary CLL cells. Any modifications on the side chain, or the rigid binding domain of the PatA macrolide ring led to complete loss of activity. Rather, introduction of an amino group at either the C2 or C3 positions of the flexible region of the macrocycle retained the activity and reduced plasma protein binding, likely through a lowered lipophilicity. We identified three new leads with potent inhibition of proteins synthesis and strong CLL cytotoxicity. They also exhibited greater selectivity towards CLL cells over normal lymphocytes comparing to the parental compound PatA. To gain structural insights into the interaction of the PatA analogs with eIF4A, a homology model of the human eIF4A1 was generated using the closed conformation of the eIF4A3 structure (PDB ID: 2HYI) as a template. The predicted PatA binding site is located at the interface of the N-terminal domain and the C-terminal domain, in between the RNA and ATP binding sites. In silico docking analysis of the PatA analogs to eIF4A correlated with their structure-activity relationships and suggested that these compounds may act by stabilizing the closed conformation of eIF4A. Thus, these novel PatA analogs hold promise for application to cancers within the appropriate biological context, such as CLL. Citation Format: Rong Chen, Mingzhao Zhu, Rajan R. Chaudhari, Omar Robles, Yuling Chen, Wesley Skillern, Qun Qin, William Wierda, Shuxing Zhang, Kenneth G. Hull, Daniel Romo, William Plunkett. Novel pateamine analogs to target the translation initiation factor eIF4A in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1854.

Co-operation of ABT-199 and gemcitabine in impeding DNA damage repair and inducing cell apoptosis for synergistic therapy of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia with limited therapeutic options available. Here, we evaluated the therapeutic potential of the combination of the Bcl-2 antagonist ABT-199 and cytotoxic agent gemcitabine in T-ALL cell lines. Our results showed that the combination of ABT-199 and gemcitabine exhibited synergistic cytotoxicity and induced significant apoptosis in human T-ALL cell lines (Jurkat and Molt4). The augmented apoptosis induced by combination treatment was accompanied by the greater extent of mitochondrial depolarization and enhanced DNA damage. Importantly, single agent induced DNA damage alone but did not inhibit RAD51/BRCA1-mediated repair for DNA double-strand breaks. In contrast, the combination of ABT-199 and gemcitabine disrupted RAD51/BRCA1-dependent DNA repair and remarkably activated caspase-3 and PARP to trigger apoptosis. Moreover, ABT-199 exerted an antagonistic action towards Bcl-2 and Bcl-xL, but to a certain extent moderately increased Mcl-1 level that could be compromised by gemcitabine. In conclusion, our study showed that the combination of ABT-199 and gemcitabine exhibited synergistic cytotoxicity in T-ALL cells by cooperatively targeting DNA damage repair pathway and Bcl-2 family proteins.

Creating novel translation inhibitors to target pro-survival proteins in chronic lymphocytic leukemia.

The viability of chronic lymphocytic leukemia (CLL) is critically dependent upon staving off death by apoptosis, a hallmark of CLL pathophysiology. The recognition that Mcl-1, a major component of the anti-apoptotic response, is intrinsically short-lived and must be continually resynthesized suggested a novel therapeutic approach. Pateamine A (PatA), a macrolide marine natural product, inhibits cap-dependent translation by binding to the initiation factor eIF4A. In this study, we demonstrated that a synthetic derivative of PatA, des-methyl des-amino PatA (DMDAPatA), blocked mRNA translation, reduced Mcl-1 protein and initiated apoptosis in CLL cells. This action was synergistic with the Bcl-2 antagonist ABT-199. However, avid binding to human plasma proteins limited DMDAPatA potency, precluding further development. To address this, we synthesized a new series of PatA analogs and identified three new leads with potent inhibition of translation. They exhibited less plasma protein binding and increased cytotoxic potency toward CLL cells than DMDAPatA, with greater selectivity towards CLL cells over normal lymphocytes. Computer modeling analysis correlated their structure-activity relationships and suggested that these compounds may act by stabilizing the closed conformation of eIF4A. Thus, these novel PatA analogs hold promise for application to cancers within the appropriate biological context, such as CLL.

Abstract 16647: Loss of Reversibility in Flow-Induced Pulmonary Arterial Hypertension is Associated With a Senescent Vascular Phenotype and Can Be Rescued by Targeted Senolysis

Introduction: Patients with congenital heart disease (CHD) are at risk of developing progressive pulmonary arterial hypertension (PAH). Timely hemodynamic unloading (HU) by surgical shunt correction usually reverses the arteriopathy, but the timepoint when or reason why PAH becomes irreversible remains elusive. Methods: PAH was created in syngeneic rats by monocrotalin (MCT) injection and an aorto-caval shunt. For HU at different PAH stages, the left donor lung with PAH was transplanted into healthy recipients at t14/t21/t28. Recipients were sacrificed 21days after transplantation. Results: MCT+Shunt induced medial neomuscularization from t14, and neointimal lesions from t21, causing arteriolar occlusion and PAH. HU normalized the vascular morphology in all t14 rats and in 7/10 t21 rats, whereas no reversal or progressive occlusion occurred in 3/10 t21 and all t28 rats. RNA-sequencing revealed that activated pro-proliferative/regenerative, pro-apoptotic and DNA-repair pathways were associated with reversal after HU. This was confirmed immunohistochemically by increased vascular-specific Ki67, Caspase3 and Rad51 protein expression. In contrast, irreversibility was associated with deactivation of latter pathways and activated pro-survival, pro-senescence and pro-inflammatory pathways, as confirmed by increased Survivin, p16 ink4A , p21 cip1 , TNFα, IL6 and MMP2. These findings were also confirmed in human PAH-CHD explant tissue. Finally, we report that the senolytic BCL2 antagonist ABT263 selectively targets rat endothelial cells made senescent by irradiation. Moreover, low-dose ABT263 treatment from t25 to t32 in MCT+Shunt rats significantly improved pulmonary hemodynamics and relieved PAH symptoms. Conclusions: Similar to clinical observations in PAH-CHD, this study showed that HU reverses PAH only in an early stage, and describes a zone of no return. We characterized the mechanistic basis for (loss of) reversibility, showing a distinct switch from increased proliferation/regeneration, apoptosis and DNA-repair, to a growth arrested, apoptosis-resistant and intrinsically pro-inflammatory phenotype, suggesting a role for senescence. Reversal of this phenotype by targeted senolysis also reversed PAH beyond the zone of no return.

Abstract 5067: BET protein proteolysis targeting chimera (BETP-PROTACs) exert more potent activity than BETP bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm (MPN) secondary (s) AML cells

Abstract In BCR-ABL1-negative myeloproliferative neoplasms with myelofibrosis (MPN-MF) transformation to AML (sAML) occurs in up to 20% of patients. Ruxolitinib (R) is a type I, ATP-competitive, JAK1 &amp; 2 inhibitor (JAKi), which is effective in the therapy of MPN-MF but does not significantly impact the clinical outcome in post-MPN sAML. We have previously reported that treatment with BETi, e.g. JQ1 or OTX015 inhibits growth and induces apoptosis of cultured sAML cells, including those that express JAK2 V617F and mutant TP53, e.g. HEL92.1.7 and SET2, as well as patient-derived (PD) CD34+ sAML cells. BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, while concomitantly inducing the levels of HEXIM1, p21, NOXA and BIM in the sAML cells. However, treatment with BETi leads to the accumulation of BETP, e.g. BRD4, which may reduce BETi-mediated repression of c-MYC, NFκB and BETP-regulated oncoproteins. In contrast, BETP-PROTACs (proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas Inc.) degrade BETPs (including BRD4) in the cultured and PD CD34+ sAML cells. At equimolar concentrations, BETP-PROTACs were significantly more potent than the BETi in inducing apoptosis of cultured and PD sAML cells (p &amp;lt; 0.05), while sparing the CD34+ normal hematopoietic progenitor cells. Notably, BETP-PROTACs caused efficient and prolonged depletion of the levels of BETPs, including BRD4 (&amp;gt; 90%) in the sAML cells. BETP-PROTAC treatment caused more up and down regulation of mRNA and protein expressions than BETi, as determined by RNA-Seq and reversed phase protein array (RPPA) analyses, respectively. As compared to treatment with BETi, BETP-PROTAC caused greater depletion of c-MYC, JAK2, p-STAT5, STAT5, p-STAT3, STAT3, PIM1 and Bcl-xL, whereas the protein levels of p21 and p27 were upregulated. CyTOF or mass-cytometry also showed that BETP-PROTAC, more than OTX015 treatment, reduced BRD4, c-MYC and p-Rb, while inducing p21 levels in the CD34+ sAML stem/progenitor cells expressing CD90, CD244, CD123 and TIM3-Fc. Compared to treatment with each agent alone, co-treatment with BETP-PROTAC and R was synergistically lethal against the cultured and PD CD34+ sAML cells. Additionally, co-treatment with BETP-PROTAC and HSP90 inhibitor AUY922 or BCL2/BcL-xL antagonist ABT263 was synergistically lethal against R-sensitive and R-resistant sAML cells. As compared to treatment with vehicle control, R treatment alone, treatment with BETP-PROTAC ARV-771 alone or in combination with R significantly reduced the in vivo sAML burden and improved the median survival of the immune-depleted mice engrafted with luciferase-transduced HEL92.1.7 cells. These findings strongly support further in vivo development of the novel BETP-PROTACs-based combinations against post-MPN sAML. Note: This abstract was not presented at the meeting. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Kanak Raina, Taghi Manshouri, Kevin G. Coleman, Yimin Qian, Andrew P. Crew, Angela Shen, Christopher P. Mill, Baohua Sun, Misun Kim, Agnieszka J. Nowak, Srdan Verstovsek, Craig M. Crews, Kapil N. Bhalla. BET protein proteolysis targeting chimera (BETP-PROTACs) exert more potent activity than BETP bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm (MPN) secondary (s) AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5067. doi:10.1158/1538-7445.AM2017-5067

Abstract B21: Novel pateamine A analogs to target pro-survival proteins in chronic lymphocytic leukemia

Abstract Introduction: Dysregulation of protein translation is a common feature of cancer. Because the great majority of oncoproteins turn-over rapidly, thus have an acute need for mRNA translation compared to housekeeping proteins, protein synthesis is a promising avenue to explore for targeted cancer therapy. The marine natural product pateamine A (PatA) inhibits cap-dependent translation initiation by binding to the eukaryotic initiation factor 4A (eIF4A). We have previously synthesized a family of PatA-based small molecules. The lead compound, des-methyl, des-amino pateamine A (DMDAPatA), is more stable and easier to synthesize than the parent natural product, and demonstrated anti-cancer activities in both cell lines and animal models. In this study, we extended the investigations of the activities of DMDAPatA in B cell malignancies, focusing on the primary chronic lymphocytic leukemia (CLL) cells. We propose to target the biological rewiring of the CLL cells, namely the overexpression of short-lived pro-survival proteins that keep these cells from undergoing apoptosis. We hypothesize that a transient inhibition of translation will cause a lethal decrease in those labile pro-survival proteins in CLL cells that will initiate apoptosis. Experimental Procedures: Primary CLL cells were used to screen the PatA analogs for their anti-leukemia activity. Cell viability was analyzed by flow cytometry. Protein translation was measured by tritium labeled leucine incorporation. Protein expression was determined by immunoblotting. Results: Our data showed that DMDAPatA blocked protein synthesis in the CLL cells. This action reduced the short-lived anti-apoptotic proteins Mcl-1 and XIAP, and initiated apoptosis in as short as 4 hr. The levels of the more stable anti-apoptotic proteins Bcl-2 and Bcl-XL were not affected. This is consistent with the selective activity of translation inhibitors toward proteins with rapid turn-over rates. Since DMDAPatA depleted Mcl-1 without affecting Bcl-2, and the Bcl-2 antagonist ABT-199 inhibits Bcl-2 activity but spares Mcl-1, the combination of DMDAPatA and ABT-199 targeted both arms of apoptosis control and killed CLL cells synergistically. Dose reduction index analysis demonstrated mutual potentiation of ABT-199 and DMDAPatA. However, in the presence of human plasma proteins, DMDAPatA lost its potency substantially. A plasma protein binding analysis showed over 99% binding in human plasma, which may hamper its development as a therapeutic agent. To address this issue, we synthesized a new series of PatA analogues with the goal of improving the physical properties and CLL potency. We found that any alteration on the binding domain of PatA, either on eastern half of the macrolide ring or the side chain, resulted in dramatic loss of activity toward the CLL cells. The scaffolding domain on the western side of the ring was more tolerable to modifications which made it feasible to improvement. We proposed to introduce an amino group at either the C2 (alpha-amino variant) or C3 position (beta-amino variant) of the macrolide ring to lower lipophilicity and reduce binding to plasma proteins. This strategy led to the identification of three new leads with potent inhibition of protein synthesis. They exhibited greater cytotoxic potency toward CLL cells than DMDAPatA and lower human plasma protein binding. They also showed better selectivity towards CLL cells over normal lymphocytes compared to the parental compound PatA, demonstrating improved therapeutic index. Conclusions: We have successfully synthesized three new PatA analogs with potent anti-leukemia activities. These compounds hold promise for application to cancers with the right biological context, such as CLL, in which the leukemia cells are addicted to the sustained expression of short-lived oncoproteins for survival. Citation Format: Rong Chen, Mingzhao Zhu, Yuling Chen, Wesley Skillern, Qun Qin, William G. Wierda, Kenneth G. Hull, Daniel Romo, William Plunkett. Novel pateamine A analogs to target pro-survival proteins in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B21.

Superior Lethal Activity of Novel BET Protein Proteolysis Targeting Chimera (BETP-PROTACs) Versus Betp Bromodomain Inhibitor (BETi) Against Post-Myeloproliferative Neoplasm (MPN) Secondary (s) AML Cells

Hematopoietic stem/progenitor cells (HPCs) of BCR-ABL1-negative myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit mutations in JAK2, c-MPL, or calreticulin (CALR) gene and display constitutive activation of JAK-STAT signaling. In MPN-MF, transformation to AML (sAML) occurs in up to 20% of patients. Ruxolitinib (R) is a type I, ATP-competitive, JAK1 & 2 inhibitor (JAKi), which is currently used in the therapy of MPN-MF. Treatment with R confers notable clinical benefit in MPN-MF, but exhibits only modest activity and does not significantly impact the clinical outcome in post-MPN sAML. Prolonged exposure to R may also lead to a loss of response, causing the emergence of JAKi-resistant (JIR) sAML cells. Although they lack additional JAK2 mutations, JIR cells exhibit reactivation of JAK-STAT signaling due to trans-phosphorylation of JAK2 by JAK1 or TYK2 tyrosine kinases (TK). Sequential genomic assessments in pre- and post-sAML transformation have revealed mutations in TET2, ASXL1, IDH1&2, SRSF2, RUNX1, MYC, PTPN11, NRAS, SETBP1 and TP53 genes. Here, we determined that treatment with BETi, e.g., JQ1 or OTX015, which disrupts the binding of BRD4 with acetylated chromatin, significantly inhibits growth and induces apoptosis of JAKi-sensitive cultured, including those that express JAK2 V617F and mutant TP53, e.g., HEL92.1.7 and SET2, and patient derived (PD) CD34+ sAML cells. Western analyses revealed that BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, while concomitantly inducing the levels of HEXIM1, p21, NOXA and BIM in the sAML cells. Co-treatment with BETi and R synergistically induced apoptosis of cultured and PD CD34+ sAML cells. As compared to treatment with vehicle control, JQ1 or R treatment alone, co-treatment with JQ1 and R significantly improved the median survival of the immune-depleted mice engrafted with HEL92.1.7 cells. However, treatment with BETi leads to the accumulation of BRD4, which could promote the activity of c-MYC, NFkB and several other transcription factors. Therefore, we compared the anti-sAML activity of the novel BETP-PROTACs (BET protein-proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas Inc.), which degrade BETPs (including BRD4), with the BETi OTX015, against cultured and PD CD34+ sAML cells. ARV-825 and ARV-771 recruit and utilize the E3 ubiquitin ligase activities of cereblon and VHL, respectively, to effectively degrade BETPs. At equimolar concentrations (100 to 500 nM) ARV-825 and ARV-771 were significantly more potent than the BETi OTX015 in inducing apoptosis of cultured and primary sAML cells (p < 0.05), while sparing the CD34+ normal hematopoietic progenitor cells. Notably, whereas OTX015 treatment increased BRD4 levels, BETP-PROTACs caused efficient and prolonged depletion of the levels of BETPs, including BRD4 (> 90%) in the sAML cells. BETP-PROTAC treatment caused more up and down regulation of mRNA and protein expressions than BETi, utilizing the RNAseq and reversed phase protein array (RPPA) analyses, respectively. Western analyses showed that BETP-PROTAC versus BETI treatment also caused greater depletion of c-MYC, JAK2, p-STAT5, STAT5, p-STAT3, STAT3, PIM1 and Bcl-xL, whereas the protein levels of p21 and p27 were upregulated. CyTOF or mass-cytometry approach showed that BETP-PROTAC, more than OTX015 treatment, reduced BRD4, c-MYC and p-RB, while inducing p21 levels in the phenotypically characterized CD34+ sAML stem/progenitor cells, based on their high expression of CD90, CD244, CD123+ and TIM3Fc+. Compared to treatment with each agent alone, co-treatment with ARV-825 and R (100 to 1000 nM) was synergistically more lethal against the cultured and PD CD34+ sAML cells. We have isolated JAKi-resistant HEL92.1.7 cells (> 10-fold resistant to R; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAKi. Notably, compared to the parental HEL92.1.7, HEL/JIR cells were highly and collaterally sensitive to BETP-PROTAC. Additionally, co-treatment with BETP-PROTAC and HSP90 inhibitor AUY922 or BCL2/BcL-xL antagonist ABT263 is synergistically lethal against JAKi sensitive and JIR sAML cells. These findings strongly support the in vivo testing of the BETP-PROTACs alone and in combinations against post-MPN sAML. These studies are underway in our laboratory and will be presented at the ASH meetings. DisclosuresRaina:Arvinas, LLC: Employment. Coleman:Arvinas, LLC: Employment. Winkler:Arvinas, LLC: Employment. Qian:Arvinas, LLC: Employment. Crew:Arvinas, LLC: Employment. Shen:Arvinas, LLC: Employment.

Inhibition of FAK Exerts Anti-Leukemic Activity and Potentiates ABT-199-Induced Apoptosis in AML

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates cell adhesion, proliferation, stem cell functions, and cell-microenvironment communications. It is activated and/or overexpressed in many malignant cells and promotes tumor progression and metastasis. Several small molecule FAK inhibitors have been developed and some of them have reached clinical trials in solid tumors. High FAK expression was found to be associated with enhanced blast migration, increased cellularity, and poor prognosis in AML, indicating that FAK could be a potential therapeutic target in AML.We showed previously that VS-4718, a potent and selective FAK inhibitor, effectively decreased viable cell number, and also induced cell death in leukemia cell lines with variable potencies in vitro, even in AML cells co-cultured with mesenchymal stromal cells (MSCs) (ASH 2015).To further examine the effect of VS-4718 in vivo, we transplanted Molm14-GFP/Luc cells into NSGS (NOD-SCID IL2Rgnull-3/GM/SF, NSG-SGM3) mice, and treated the mice with VS-4718 (75 mg/kg) twice a day via oral gavage. We found that VS-4718 as a single agent exerted anti-leukemia activity as assessed by in vivo imaging for leukemia burden, human CD45 positivity in mouse peripheral blood, and histological staining of mouse tissues. VS-4718 treated mice survived significantly longer than the untreated controls (medium survival 27 vs 20 days, P = 0.0003).FAK activates multiple signaling pathways and supports tumor cell survival. We found that inhibition of FAK with VS-4718 in Molm14 cells reduced the expression of MCL-1. The BCL-2 antagonist ABT-199 is being tested clinically for the treatment of hematological malignancies. However, as a single agent, ABT-199-treated cells can acquire drug resistance by upregulating MCL-1 and BCL-XL after treatment. We therefore hypothesized that combination of VS-4718 and ABT-199 would be more effective in inducing cell death and reversing the resistance of AML cells exposed to ABT-199 alone. In vitro studies showed that VS-4718 significantly improved the potency of ABT-199 in AML cell lines (ABT-199 EC50 at 24 h: 880.3 nM and 14.5 nM in the presence of 0.4 mM VS-4718, respectively, in Molm14 cells), and the combination of VS-4718 and ABT-199 also synergistically killed primary AML cells even when co-cultured with MSCs in the majority of samples examined, while largely sparing normal BM CD34+ cells.Furthermore, the upregulation of MCL-1 in ABT-199-treated AML cells was antagonized by combining ABT-199 with VS-4718. BCL-XL is known to be regulated by STAT5. The activation of STAT5, which can be regulated by FAK, is considered to be significant in maintaining MCL-1 expression in FLT3-ITD AML cells. We observed that treatment with VS-4718 decreased the level of p-STAT5 as well as MCL-1 and BCL-XL in Molm14 cells harboring FLT3-ITD mutation.These results suggest a novel therapeutic strategy for targeting FAK and BCL-2 family proteins for the treatment of AML. DisclosuresPachter:Verastem, Inc: Employment. Weaver:Verastem, Inc: Employment. Carter:PRISM Pharma/Eisai: Research Funding.

Abstract 3557: System-based BCL2 family protein signatures as predictive biomarkers in triple-negative breast cancer

Abstract Breast cancer is a heterogeneous disease. More recently, the molecular basis for this heterogeneity has started to be analysed at the genomic, transcriptomic and proteomic level, allowing for the development of more personalized therapies. Cancer cells showing resistance to therapy frequently have an intrinsic deficiency in their ability to initiate or execute apoptosis [1]. The BCL2 (B-cell lymphoma 2) family of proteins controls the process of Mitochondrial Outer Membrane Permeabilization (MOMP), which is required for the activation of the mitochondrial apoptosis pathway [2]. We developed a deterministic systems model of BCL2 protein interactions, DR_MOMP that calculates the sensitivity of cells to undergo mitochondrial apoptosis [3]. Here we applied DR_MOMP in the context of triple negative breast cancer to determine whether this systems model can be used as a prognostic biomarker or patient stratification tool. To analyze the contribution of BCL2 proteins in modulating apoptosis in breast cancer we validated DR_MOMP in a panel of Triple Negative Breast Cancer (TNBC) cell lines. Using quantitative Western blotting we determined the absolute protein levels of pro-apoptotic BAX and BAK and anti-apoptotic BCL2, BCL(X)L and MCL1. We found a significant correlation between cell survival and BCL(X)L levels (ρ = 0.76) after Cisplatin/Paclitaxel treatment, but protein levels of BCL2, MCL-1, BAX and BAK did not correlate with cell survival. Using absolute protein levels as input for DR_MOMP, we found a strongly improved correlation between model predictions and cells’ responses to Cisplatin (ρ = 0.93) and Paclitaxel treatments (ρ = 0.97). Next we performed synergy studies using the selective BCL2 antagonist ABT-199 or the selective BCL(X)L antagonist WEHI-539 in combination with cisplatin. We observed differential effects of BCL(X)L and BCL-2 inhibition in individual cell lines that were remodelled by DR_MOMP. Modelling also suggested additional effects of BCL2 antagonists independent of MOMP regulation. BCL2 profiling was also performed in primary tumours of 16 TBNC patients in order to predict the patients’ risk prospectively. DR_MOMP predicted a high risk in 37.5% of patients which is in line with 5-year recurrence rates in the literature [4]. Our findings provide evidence that DR_MOMP can be deployed to predict the response of TNBC breast cancer to genotoxic therapy, and can aid in the choice of the optimal BCL2 antagonists. This work is supported by Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT [1] Pillai et al. Am J Cancer Res. 2013 Jun 20;3(3):312-22. PubMed PMID: 23841030. [2] Lee et al. BMC Cancer. 2007 Apr 12;7:63. PubMed PMID: 17430582. [3] Lindner et al. Cancer Res. 2013 Jan 15;73(2):519-28. PubMed PMID: 23329644 [4] Haffty et al. J Clin Oncol. 2006 Dec 20;24(36):5652-7. PubMed PMID: 17116942 Citation Format: Federico Lucantoni, Andreas U. Lindner, Norma O’Donovan, Damir Vareslija, Lance Hudson, Arnold DK Hill, Michael Kerin, Roisin Dwyer, Leonie Young, Jochen HM Prehn. System-based BCL2 family protein signatures as predictive biomarkers in triple-negative breast cancer. [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 3557.

BCR signaling inhibitors differ in their ability to overcome Mcl-1–mediated resistance of CLL B cells to ABT-199

AbstractThe Bcl-2 antagonist ABT-199 (venetoclax) has demonstrated promising clinical activity in patients with chronic lymphocytic leukemia (CLL). ABT-199 is strongly cytotoxic against unstimulated peripheral blood CLL cells in vitro but is much less effective against CLL cells that have received survival signals from the microenvironment. In particular, stimulation of CLL cells with CD40L results in substantial resistance mediated by induction of the antiapoptotic Bcl-2 family proteins Bcl-xL and Bfl-1. In this study, we investigated whether resistance to ABT-199 can be conferred by B-cell receptor (BCR) stimulation, which is another important survival signal from the leukemic microenvironment. We show that sustained BCR stimulation results in significant ABT-199 resistance, which correlates with induction of the antiapoptotic protein Mcl-1 and less consistently with downregulation of proapoptotic Bmf, Hrk, and BimEL. A major role for Mcl-1 in conferring ABT-199 resistance is additionally supported by knockdown and enforced expression experiments with primary CLL cells. We further show that SYK, BTK, and phosphatidylinositol 3-kinase δ (PI3Kδ) inhibitors significantly downregulate Mcl-1, but with different efficacy. Complete Mcl-1 downregulation was consistently achieved only with SYK inhibitors R406 and GS-9973 (entospletinib), whereas the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib in more than half of the cases had only a partial effect. The greater ability of SYK inhibitors to downregulate Mcl-1 correlated with their greater capacity to block BCR-mediated inactivation of GSK-3, a major negative regulator of Mcl-1. The finding that BCR signaling inhibitors differ in their ability to target Mcl-1 is relevant for the design of clinical trials combining these agents with ABT-199.