Joel D. Leverson

ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets

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-XL), which has shown clinical efficacy in some BCL-2–dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-XL 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.

Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins

The function of small ubiquitin‐like modifier (SUMO)‐binding proteins is key to understanding how SUMOylation regulates cellular processes. We identified two related Schizosaccharomyces pombe proteins, Rfp1 and Rfp2, each having an N‐terminal SUMO‐interacting motif (SIM) and a C‐terminal RING‐finger domain. Genetic analysis shows that Rfp1 and Rfp2 have redundant functions; together, they are essential for cell growth and genome stability. Mammalian RNF4, an active ubiquitin E3 ligase, is an orthologue of Rfp1/Rfp2. Rfp1 and Rfp2 lack E3 activity but recruit Slx8, an active RING‐finger ubiquitin ligase, through a RING–RING interaction, to form a functional E3. RNF4 complements the growth and genomic stability defects of rfp1rfp2, slx8, and rfp1rfp2slx8 mutant cells. Both the Rfp‐Slx8 complex and RNF4 specifically ubiquitylate artificial SUMO‐containing substrates in vitro in a SUMO binding‐dependent manner. SUMOylated proteins accumulate in rfp1rfp2 double‐null cells, suggesting that Rfp/Slx8 proteins may promote ubiquitin‐dependent degradation of SUMOylated targets. Hence, we describe a family of SIM‐containing RING‐finger proteins that potentially regulates eukaryotic genome stability through linking SUMO‐interaction with ubiquitin conjugation.

Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo

The Akt kinases are central nodes in signal transduction pathways that are important for cellular transformation and tumor progression. We report the development of a series of potent and selective indazole-pyridine based Akt inhibitors. These compounds, exemplified by A-443654 (Ki = 160 pmol/L versus Akt1), inhibit Akt-dependent signal transduction in cells and in vivo in a dose-responsive manner. In vivo, the Akt inhibitors slow the progression of tumors when used as monotherapy or in combination with paclitaxel or rapamycin. Tumor growth inhibition was observed during the dosing interval, and the tumors regrew when compound administration was ceased. The therapeutic window for these compounds is narrow. Efficacy is achieved at doses ∼2-fold lower than the maximally tolerated doses. Consistent with data from knockout animals, the Akt inhibitors induce an increase in insulin secretion. They also induce a reactive increase in Akt phosphorylation. Other toxicities observed, including malaise and weight loss, are consistent with abnormalities in glucose metabolism. These data show that direct Akt inhibition may be useful in cancer therapy, but significant metabolic toxicities are likely dose limiting.

Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy

Selective inhibition of BCL-XL synergizes with docetaxel to inhibit the growth of solid tumors but does not inhibit granulopoiesis. A more refined antitumor strategy The BCL-2 family is a group of related proteins that regulate apoptosis in a variety of ways. The success of anticancer treatments often hinges on the ability to induce cancer cell death by apoptosis. As a result, there has been a great deal of interest in developing drugs that can inhibit the antiapoptotic members of the BCL-2 pathway. Unfortunately, some of these drugs are also associated with dose-limiting hematologic toxicities, such as neutropenia. Now, Leverson et al. have used a toolkit of BCL-2 family inhibitors with different specificities to show that specifically inhibiting BCL-XL (one member of this protein family) is effective for killing tumors, but without the common side effects seen with less selective drugs. 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.

Efficacy and Biological Correlates of Response in a Phase II Study of Venetoclax Monotherapy in Patients with Acute Myelogenous Leukemia

We present a phase II, single-arm study evaluating 800 mg daily venetoclax, a highly selective, oral small-molecule B-cell leukemia/lymphoma-2 (BCL2) inhibitor in patients with high-risk relapsed/refractory acute myelogenous leukemia (AML) or unfit for intensive chemotherapy. Responses were evaluated following revised International Working Group (IWG) criteria. The overall response rate was 19%; an additional 19% of patients demonstrated antileukemic activity not meeting IWG criteria (partial bone marrow response and incomplete hematologic recovery). Twelve (38%) patients had isocitrate dehydrogenase 1/2 mutations, of whom 4 (33%) achieved complete response or complete response with incomplete blood count recovery. Six (19%) patients had BCL2-sensitive protein index at screening, which correlated with time on study. BH3 profiling was consistent with on-target BCL2 inhibition and identified potential resistance mechanisms. Common adverse events included nausea, diarrhea and vomiting (all grades), and febrile neutropenia and hypokalemia (grade 3/4). Venetoclax demonstrated activity and acceptable tolerability in patients with AML and adverse features. SIGNIFICANCE Venetoclax monotherapy demonstrated clinical activity in patients with AML (relapsed/refractory or unfit for intensive chemotherapy) with a tolerable safety profile in this phase II study. Predictive markers of response consistent with BCL2 dependence were identified. Clinical and preclinical findings provide a compelling rationale to evaluate venetoclax combined with other agents in AML. Cancer Discov; 6(10); 1106-17. ©2016 AACRSee related commentary by Pullarkat and Newman, p. 1082This article is highlighted in the In This Issue feature, p. 1069.

Small Interfering RNA–Mediated Polo-Like Kinase 1 Depletion Preferentially Reduces the Survival of p53-Defective, Oncogenic Transformed Cells and Inhibits Tumor Growth in Animals

Polo-like kinase 1 (Plk1) is required for multiple stages of mitosis and is up-regulated in many human malignancies. We depleted Plk1 expression using small interfering RNA (siRNA) and showed defects in bipolar spindle formation and cytokinesis, growth inhibition, and apoptosis induction in human cancer cell lines. To our surprise, depletion of Plk1 in normal human cells did not result in obvious cell cycle defects, and did not induce significant inhibition of cell growth for at least two cell cycles. In addition, Plk1 siRNA inhibited colony formation in soft agar and tumorigenesis in a HT1080 xenograft model in a dose-dependent manner. Analysis with isogenic pairs of cell lines, differing in p53 status, revealed that Plk1 depletion preferentially induced mitotic arrest, aneuploidy, and reduced cell survival in the p53-defective cell lines. No obvious defects were observed in most p53 wild-type cells during the first few cell cycles. In addition, long-term survival studies revealed that p53 facilitates survival upon Plk1 depletion. Therefore, short-term inhibition of Plk1 can kill tumor cells while allowing normal cells to survive. These data validate the episodic inhibition of Plk1 as a very useful approach for cancer treatment.

Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition.

Rapamycin, a natural product inhibitor of the Raptor-mammalian target of rapamycin complex (mTORC1), is known to induce Protein kinase B (Akt/PKB) Ser-473 phosphorylation in a subset of human cancer cell lines through inactivation of S6K1, stabilization of insulin receptor substrate (IRS)-1, and increased signaling through the insulin/insulin-like growth factor-I/phosphatidylinositol 3-kinase (PI3K) axis. We report that A-443654, a potent small-molecule inhibitor of Akt serine/threonine kinases, induces Akt Ser-473 phosphorylation in all human cancer cell lines tested, including PTEN- and TSC2-deficient lines. This phenomenon is dose-dependent, manifests coincident with Akt inhibition and likely represents an alternative, rapid-feedback pathway that can be functionally dissociated from mTORC1 inhibition. Experiments performed in TSC2−/− cells indicate that TSC2 and IRS-1 cooperate with, but are dispensable for, A-443654-mediated Akt phosphorylation. This feedback event does require PI3K activity, however, as it can be inhibited by LY294002 or wortmannin. Small interfering RNA-mediated knockdown of mTOR or Rictor, components of the rapamycin-insensitive mTORC2 complex, but not the mTORC1 component Raptor, also inhibited Akt Ser-473 phosphorylation induced by A-443654. Our data thus indicate that Akt phosphorylation and activity are coupled in a manner not previously appreciated and provide a novel mode of Akt regulation that is distinct from the previously described rapamycin-induced IRS-1 stabilization mechanism.

From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors

Members of the B cell lymphoma 2 (BCL-2) gene family have a central role in regulating programmed cell death by controlling pro-apoptotic and anti-apoptotic intracellular signals. In cancer, apoptosis evasion through dysregulation of specific BCL-2 family genes is a recurring event; accordingly, selective inhibition of specific anti-apoptotic BCL-2 family proteins represents an exciting therapeutic opportunity. A combination of nuclear magnetic resonance (NMR)-based screening and structure-based drug design has yielded the first bona fide BCL-2 homology 3 (BH3) mimetics, including the BCL-2 and BCL-XL dual antagonist navitoclax, which is the first BCL-2 family inhibitor to show efficacy in patients with cancer. Clinical experience with navitoclax prompted the generation of the highly selective BCL-2 inhibitor venetoclax, which is now approved in the United States for the treatment of patients with chronic lymphocytic leukaemia with 17p deletion who have received at least one prior therapy. Recent advances have also been made in the development of potent and selective inhibitors of BCL-XL and myeloid cell leukaemia 1 (MCL1), which are additional BCL-2 family members with established anti-apoptotic roles in cancer. Here we review the latest progress in direct and selective targeting of BCL-2 family proteins for cancer therapy.

Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity

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.

Mcl-1 is critical for survival in a subgroup of non-small-cell lung cancer cell lines

Non-small-cell lung cancer (NSCLC) is the most deadly type of cancer in the United States and worldwide. Although new therapy is available, the survival rate of NSCLC patients remains low. One hallmark of cancer cells is defects in the apoptotic cell death program. In this study, we investigate the role of B-cell lymphoma 2 (Bcl-2) family members Bcl-2, Bcl-xL and Mcl-1, known to regulate cell survival and death, in a panel of fourteen NSCLC cell lines. NSCLC cell lines express high levels of Mcl-1 and Bcl-xL, but not Bcl-2. Silencing the expression of Mcl-1 with small interfering RNA (siRNA) oligonucleotides potently killed a subgroup of NSCLC cell lines. In contrast, Bcl-xL siRNA had no effect in these lines unless Mcl-1 siRNA was also introduced. Interestingly, high MCL1 to BCL-xl messenger RNA determines whether the cells depend on Mcl-1 for survival. We further investigated the role of Mcl-1 in NSCLC cells using a Mcl-1-dependent cell line, H23. The expression of a complementary DNA containing only the coding region of MCL1 rescued H23 cells from the toxicity of a 3′ untranslated region (UTR) targeting Mcl-1 siRNA but not a siRNA targeting the coding region of MCL1. Furthermore, we show that Mcl-1 sequesters the BH3-only protein Noxa and Bim and the apoptotic effector Bak. Not surprisingly, Noxa, Bim, or Bak knockdown partially rescued H23 cells from toxicity mediated by Mcl-1 siRNA to different degrees. Collectively, our results indicate that targeting Mcl-1 may improve therapy for a subset of NSCLC patients.