Susan E. Morgan-Lappe

Influence of Bcl-2 Family Members on the Cellular Response of Small-Cell Lung Cancer Cell Lines to ABT-737

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.

'Seed' analysis of off-target siRNAs reveals an essential role of Mcl-1 in resistance to the small-molecule Bcl-2/Bcl-XL inhibitor ABT-737.

ABT-737 is a subnanomolar inhibitor of the antiapoptotic proteins Bcl-2, Bcl-XL and Bcl-w. Although ABT-737 triggers extensive cell death in many small-cell lung carcinoma (SCLC) cell lines, some of the SCLC cell lines and the majority of the cancer cell lines derived from other solid tumors were found to be resistant to ABT-737. To better understand the mechanism of resistance to ABT-737, we screened a short interfering RNA library consisting of short interfering RNA against 4000 ‘druggable’ targets in an SCLC-derived cell line, NCI-H196. By comparing the knockdowns with phenotypes, all of the three top ‘hits’ from the screen were found to result from off-target gene silencing. Interestingly, the three off-target siRNAs were found to knock down an antiapoptotic Bcl-2 family protein Mcl-1 owing to the complementation between their seed regions with the 3′ untranslated region (3′ UTR) of Mcl-1. Furthermore, reducing the level of Mcl-1 using siRNAs or the small-molecule compounds Bay43-9006 and Seliciclib was sufficient to overcome the resistance to ABT-737 in the resistant SCLC cell line and cancer cell lines derived from other solid tumors. These results provide further evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and the combination of Mcl-1 downregulating agents with ABT-737 could be potent therapeutic regimens for patient with ABT-737-resistant SCLC and many other types of solid tumors.

Identification of Ras-Related Nuclear Protein, Targeting Protein for Xenopus Kinesin-like Protein 2, and Stearoyl-CoA Desaturase 1 as Promising Cancer Targets from an RNAi-Based Screen

To identify new candidate cancer drug targets, we used RNAi as a tool to functionally evaluate genes that play a role in maintaining human tumor cell survival. We screened a small interfering RNA (siRNA) library directed against approximately 3,700 individual genes to assess the ability of siRNAs to induce cell death in an in vitro cell cytotoxicity assay. We found that siRNAs specifically targeting ras-related nuclear protein (Ran), targeting protein for Xenopus kinesin-like protein 2 (TPX2), and stearoyl-CoA desaturase 1 (SCD1), significantly reduced the survival of multiple human tumor cell lines. Further target validation studies revealed that treatment with Ran and TPX2 siRNAs differentially reduced the survival of activated K-Ras-transformed cells compared with their normal isogenic counterparts in which the mutant K-Ras gene had been disrupted (DKS-8). Knockdown of Ran and TPX2 in activated mutant K-Ras cells selectively induced S-phase arrest or transient G(2)-M arrest phenotypes, respectively, that preceded apoptotic cell death. Given our observations that Ran and TPX2 depletion preferentially reduces the survival of activated K-Ras-transformed cells, these two proteins may serve as useful anticancer targets in tumors expressing the activated K-Ras oncogene.

Growth Inhibition and Radiosensitization of Glioblastoma and Lung Cancer Cells by Small Interfering RNA Silencing of Tumor Necrosis Factor Receptor–Associated Factor 2

Radiotherapy combined with chemotherapy is the treatment of choice for glioblastoma and locally advanced lung cancer, but radioresistance of these two types of cancer remains a significant therapeutic hindrance. To identify molecular target(s) for radiosensitization, we screened a small interfering RNA (siRNA) library targeting all protein kinases and E3 ubiquitin ligases in the human genome and identified tumor necrosis factor receptor-associated factor 2 (TRAF2). Silencing of TRAF2 using siRNA caused a significant growth suppression of glioblastoma U251 cells and moderately sensitized these radioresistant cells to radiation. Overexpression of a really interesting new gene (RING)-deleted dominant-negative TRAF2 mutant also conferred radiosensitivity, whereas overexpression of wild-type (WT) TRAF2 significantly protected cells from radiation-induced killing. Likewise, siRNA silencing of TRAF2 in radioresistant lung cancer H1299 cells caused growth suppression and radiosensitization, whereas overexpression of WT TRAF2 enhanced radioresistance in a RING ligase-dependent manner. Moreover, siRNA silencing of TRAF2 in UM-SCC-1 head and neck cancer cells also conferred radiosensitization. Further support for the role of TRAF2 in cancer comes from the observations that TRAF2 is overexpressed in both lung adenocarcinoma tissues and multiple lung cancer cell lines. Importantly, TRAF2 expression was very low in normal bronchial epithelial NL20 cells, and TRAF2 silencing had a minimal effect on NL20 growth and radiation sensitivity. Mechanistically, TRAF2 silencing blocks the activation of the nuclear factor-kappaB signaling pathway and down-regulates several G(2)-M cell cycle control proteins, resulting in enhanced G(2)-M arrest, growth suppression, and radiosensitization. Our studies suggest that TRAF2 is an attractive drug target for anticancer therapy and radiosensitization.

Survivin depletion preferentially reduces the survival of activated K-Ras-transformed cells

To identify cancer-specific targets, we have conducted a synthetic lethal screen using a small interfering RNA (siRNA) library targeting ∼4,000 individual genes for enhanced killing in the DLD-1 colon carcinoma cell line that expresses an activated copy of the K-Ras oncogene. We found that siRNAs targeting baculoviral inhibitor of apoptosis repeat-containing 5 (survivin) significantly reduced the survival of activated K-Ras-transformed cells compared with its normal isogenic counterpart in which the mutant K-Ras gene had been disrupted (DKS-8). In addition, survivin siRNA induced a transient G2-M arrest and marked polyploidy that was associated with increased caspase-3 activation in the activated K-Ras cells. These results indicate that tumors expressing the activated K-Ras oncogene may be particularly sensitive to inhibitors of the survivin protein. [Mol Cancer Ther 2007;6(1):269–76]

Kinome siRNA-phosphoproteomic screen identifies networks regulating AKT signaling.

To identify regulators of intracellular signaling, we targeted 541 kinases and kinase-related molecules with small interfering RNAs (siRNAs), and determined their effects on signaling with a functional proteomics reverse-phase protein array (RPPA) platform assessing 42 phospho and total proteins. The kinome-wide screen demonstrated a strong inverse correlation between phosphorylation of AKT and mitogen-activated protein kinase (MAPK) with 115 genes that, when targeted by siRNAs, demonstrated opposite effects on MAPK and AKT phosphorylation. Network-based analysis identified the MAPK subnetwork of genes along with p70S6K and FRAP1 as the most prominent targets that increased phosphorylation of AKT, a key regulator of cell survival. The regulatory loops induced by the MAPK pathway are dependent on tuberous sclerosis complex 2 but demonstrate a lesser dependence on p70S6K than the previously identified FRAP1 feedback loop. The siRNA screen also revealed novel bi-directionality in the AKT and GSK3 (Glycogen synthase kinase 3) interaction, whereby genetic ablation of GSK3 significantly blocks AKT phosphorylation, an unexpected observation as GSK3 has only been predicted to be downstream of AKT. This method uncovered novel modulators of AKT phosphorylation and facilitated the mapping of regulatory loops.

RNAi-based screening of the human kinome identifies Akt-cooperating kinases: a new approach to designing efficacious multitargeted kinase inhibitors.

Tumors comprise genetically heterogeneous cell populations, whose growth and survival depend on multiple signaling pathways. This has spurred the development of multitargeted therapies, including small molecules that can inhibit multiple kinases. A major challenge in designing such molecules is to determine which kinases to inhibit in each cancer to maximize efficacy and therapeutic index. We describe an approach to this problem implementing RNA interference technology. In order to identify Akt-cooperating kinases, we screened a library of kinase-directed small interfering RNAs (siRNAs) for enhanced cancer cell killing in the presence of Akt inhibitor A-443654. siRNAs targeting casein kinase I gamma 3 (CSNK1G3) or the inositol polyphosphate multikinase (IPMK) significantly enhanced A-443654-mediated cell killing, and caused decreases in Akt Ser-473 and ribosomal protein S6 phosphorylation. Small molecules targeting CSNK1G3 and/or IPMK in addition to Akt may thus exhibit increased efficacy and have the potential for improved therapeutic index.

LRRC15 is a novel mesenchymal protein and stromal target for antibody-drug conjugates.

Progress in understanding tumor stromal biology has been constrained in part because cancer-associated fibroblasts (CAF) are a heterogeneous population with limited cell-type-specific protein markers. Using RNA expression profiling, we identified the membrane protein leucine-rich repeat containing 15 (LRRC15) as highly expressed in multiple solid tumor indications with limited normal tissue expression. LRRC15 was expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 expression was induced by TGFβ on activated fibroblasts (αSMA+) and on mesenchymal stem cells. These collective findings suggested LRRC15 as a novel CAF and mesenchymal marker with utility as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate (ADC) directed against LRRC15, and it demonstrated robust preclinical efficacy against LRRC15 stromal-positive/cancer-negative, and LRRC15 cancer-positive models as a monotherapy, or in combination with standard-of-care therapies. ABBV-085s unique mechanism of action relied upon the cell-permeable properties of MMAE to preferentially kill cancer cells over LRRC15-positive CAF while also increasing immune infiltrate (e.g., F4/80+ macrophages) in the tumor microenvironment. In summary, these findings validate LRRC15 as a novel therapeutic target in multiple solid tumor indications and support the ongoing clinical development of the LRRC15-targeted ADC ABBV-085.Significance: These findings identify LRRC15 as a new marker of cancer-associated fibroblasts and cancers of mesenchymal origin and provide preclinical evidence for the efficacy of an antibody-drug conjugate targeting the tumor stroma. Cancer Res; 78(14); 4059-72. ©2018 AACR.

ABT-165, a Dual Variable Domain Immunoglobulin (DVD-Ig) Targeting DLL4 and VEGF, Demonstrates Superior Efficacy and Favorable Safety Profiles in Preclinical Models

Antiangiogenic therapy is a clinically validated modality in cancer treatment. To date, all approved antiangiogenic drugs primarily inhibit the VEGF pathway. Delta-like ligand 4 (DLL4) has been identified as a potential drug target in VEGF-independent angiogenesis and tumor-initiating cell (TIC) survival. A dual-specific biologic targeting both VEGF and DLL4 could be an attractive strategy to improve the effectiveness of anti-VEGF therapy. ABT-165 was uniquely engineered using a proprietary dual-variable domain immunoglobulin (DVD-Ig) technology based on its ability to bind and inhibit both DLL4 and VEGF. In vivo, ABT-165 induced significant tumor growth inhibition compared with either parental antibody treatment alone, due, in part, to the disruption of functional tumor vasculature. In combination with chemotherapy agents, ABT-165 also induced greater antitumor response and outperformed anti-VEGF treatment. ABT-165 displayed nonlinear pharmacokinetic profiles in cynomolgus monkeys, with an apparent terminal half-life > 5 days at a target saturation dose. In a GLP monkey toxicity study, ABT-165 was well-tolerated at doses up to 200 mg/kg with non-adverse treatment–related histopathology findings limited to the liver and thymus. In summary, ABT-165 represents a novel antiangiogenic strategy that potently inhibits both DLL4 and VEGF, demonstrating favorable in vivo efficacy, pharmacokinetic, and safety profiles in preclinical models. Given these preclinical attributes, ABT-165 has progressed to a phase I study. Mol Cancer Ther; 17(5); 1039–50. ©2018 AACR.

Abstract DDT01-03: ABBV-621: A best-in-class TRAIL-receptor agonist fusion protein that enhances optimal clustering for the treatment of solid and hematologic tumors

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of proteins that play diverse roles in the activation of several intracellular signaling pathways that control cell proliferation, survival, and apoptosis. Activation of the TRAIL pathway has been viewed as an attractive therapeutic approach for the treatment of cancer because of its distinct role in the induction of tumor cell apoptosis. TRAIL can bind as a trimer to membrane-bound or soluble receptors, and only the two closely related cell surface death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) can preferentially trigger the extrinsic pathway of tumor cells, leading to apoptotic cell death. The BCL-2 family member-driven mitochondrial apoptotic pathway can also serve to amplify the extrinsic pathway through further downstream activation of caspases. Given the crosstalk between the extrinsic and intrinsic cell death pathways, several resistance mechanisms have emerged to escape TRAIL-induced apoptosis. These include altered receptor regulation, overexpression of cellular FLICE-like inhibitory protein (cFLIP) that inhibits the proapoptotic activity of the DISC, overexpression of the prosurvival BCL-2 family member proteins, and the inhibitor of apoptosis proteins (IAPs) that directly inhibit caspases. In addition, TRAIL can induce non–cell death signaling pathways (e.g., NF-κB, MAPK, PI3K) to promote tumor cell growth as a potential resistance mechanism. Results of several clinical trials that targeted DR4 and DR5 with first-generation agonistic anti-TRAIL antibodies or human recombinant TRAIL indicated that these agents failed to demonstrate significant improvement in patient progression-free survival compared to standard-of-care therapy. The major contributing factor in this lack of clinical activity is believed to be the suboptimal TRAIL receptor clustering by these agonists. ABBV-621 is a novel, second-generation TRAIL receptor agonist comprising a human IgG1-Fc linked to native single-chain TRAIL-receptor binding domain (scTRAIL-RBD) monomers that are covalently connected by glycosylated linkers, resulting in two sets of trimeric RBDs. ABBV-621 is designed to maximize receptor clustering butdoes not require Fcγ-R-mediated crosslinking for optimal in vivo efficacy, which has been deemed an activity-limiting step for the competitor anti-DR4 and anti-DR5 antibodies in the clinic. In-house studies have demonstrated the requirement for enhanced caspase-8 aggregation into the death-inducing signaling complex (DISC) to induce potent tumor cell death, providing mechanistic insights as to why death receptor agonists originally failed and further distinguishing ABBV-621 from the first-generation agents. ABBV-621 induces dose-dependent apoptotic cell death at sub- to single-digit nanomolar potencies across a large panel (~100) of human hematologic and solid tumor cell lines in vitro. ABBV-621 activity is on-target and mechanism-based as demonstrated by a rapid activation of downstream apoptotic signaling events (cleavage of caspase-8, caspase-3, and PARP). In human tumor xenograft models, ABBV-621 exhibits potent antitumor activity in vivo as a monotherapy and in combination with targeted agents or chemotherapy using xenograft tumors derived from colorectal, lung, leukemia, and lymphoma cell lines. This activity is on-target, mechanism-based, and dose-dependent as indicated by rapid activation of tumor caspase-8 and caspase-3 after a single ascending dose of ABBV-621 in a colorectal carcinoma xenograft model. ABBV-621 exhibits a dose-proportional pharmacokinetic (PK) profile in mice and cynomolgus monkeys, and the projected human half-life is estimated to be ~2 days. PK/pharmacodynamic (PD) assessments estimate that achieving exposure above an efficacious concentration for 4-7 days is sufficient to maintain tumor regressions up to 4-7 weeks. Screens against 95 patient-derived xenograft (PDX) models consisting of 12 different solid tumor types uncovered single-agent activity (regressions) in >50% of all PDXs tested. PDX models can be more reflective of human disease by providing estimation of effects of tumor heterogeneity on ABBV-621 activity. Models that were partially sensitive or completely resistant to treatment were also identified, and collectively these responses are being utilized with associated pre- and post-treatment genomic/proteomic information to further inform potential patient stratification markers. To demonstrate on-target biologic activity in the clinic, proximal and distal target engagement markers will be pursued. In a GLP monkey toxicity study, ABBV-621 was well tolerated with no adverse test item–related findings. The first-in-human (FIH) phase 1/1b study with ABBV-621 will enroll subjects with previously treated advanced solid tumors and hematologic malignancies. The phase 1 objectives will be to establish the safety and tolerability of ABBV-621, as well as to understand the PK properties. Secondary objectives will be to explore target engagement and efficacy biomarkers and to evaluate clinical activity. Citation Format: Susan E. Morgan-Lappe. ABBV-621: A best-in-class TRAIL-receptor agonist fusion protein that enhances optimal clustering for the treatment of solid and hematologic tumors [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 DDT01-03. doi:10.1158/1538-7445.AM2017-DDT01-03