Darcy J. P. Bates

Multiple BH3 Mimetics Antagonize Antiapoptotic MCL1 Protein by Inducing the Endoplasmic Reticulum Stress Response and Up-regulating BH3-only Protein NOXA

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-XL, or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.

Vinblastine Induces Acute, Cell Cycle Phase–Independent Apoptosis in Some Leukemias and Lymphomas and Can Induce Acute Apoptosis in Others when Mcl-1 Is Suppressed

Chemotherapeutic agents modify intracellular signaling that culminates in the inhibition of Bcl-2 family members and initiates apoptosis. Inhibition of the extracellular signal-regulated kinase by PD98059 dramatically accelerates vinblastine-mediated apoptosis in ML-1 leukemia with cells dying in 4 hours from all phases of the cell cycle. Inhibition of protein synthesis by cycloheximide also markedly accelerated vinblastine-induced apoptosis, showing that the proteins required for this acute apoptosis are constitutively expressed. Vinblastine induced the rapid induction of Mcl-1 that was inhibited by PD98059 and cycloheximide. No change in Bcl-2 or Bcl-X was observed. We hypothesize that ML-1 cells use Mcl-1 for protection from the rapid vinblastine-induced apoptosis. This was confirmed by targeting Mcl-1 with short hairpin RNA. We also investigated the response of 13 other leukemia and lymphoma cell lines and cells from seven chronic lymphocytic leukemia patients. Four cell lines and all chronic lymphocytic leukemia cells were killed in 6 hours by vinblastine alone. Two additional cell lines were sensitized to vinblastine by PD98059, which suppressed Mcl-1. This acute apoptosis either alone or in combination with PD98059 required vinblastine-mediated activation of c-Jun-NH2-terminal kinase. PD98059 did not suppress Mcl-1 in other cell lines whereas sorafenib did, but this did not sensitize the cells to vinblastine, suggesting that the acute apoptosis varies depending on which Bcl-2 protein mediates protection. Most of the cell lines were sensitized to vinblastine by cycloheximide, suggesting that inhibition of a short-lived protein in addition to Mcl-1 can acutely sensitize cells. These results suggest several clinical strategies that might provide an effective therapy for selected patients. Mol Cancer Ther; 9(4); 791–802. ©2010 AACR.

Microtubule destabilising agents: far more than just antimitotic anticancer drugs

Vinca alkaloids have been approved as anticancer drugs for more than 50 years. They have been classified as cytotoxic chemotherapy drugs that act during cellular mitosis, enabling them to target fast growing cancer cells. With the evolution of cancer drug development there has been a shift towards new “targeted” therapies to avoid the side effects and general toxicities of “cytotoxic chemotherapies” such as the vinca alkaloids. Due to their original classification, many have overlooked the fact that vinca alkaloids, taxanes and related drugs do have a specific molecular target: tubulin. They continue to be some of the most effective anticancer drugs, perhaps because their actions upon the microtubule network extend far beyond the ability to halt cells in mitosis, and include the induction of apoptosis at all phases of the cell cycle. In this review, we highlight the numerous cellular consequences of disrupting microtubule dynamics, expanding the textbook knowledge of microtubule destabilising agents and providing novel opportunities for their use in cancer therapy.

Vinblastine Rapidly Induces NOXA and Acutely Sensitizes Primary Chronic Lymphocytic Leukemia Cells to ABT-737

Proteins of the BCL2 family provide a survival mechanism in many human malignancies, including chronic lymphocytic leukemia (CLL). The BCL2 inhibitor ABT-263 (navitoclax) is active in clinical trials for lymphoid malignancies, yet resistance is expected on the basis of preclinical models. We recently showed that vinblastine can dramatically sensitize several leukemia cell lines to ABT-737 (the experimental congener of ABT-263). The goal of these experiments was to determine the impact of vinblastine on ABT-737 sensitivity in CLL cells isolated from peripheral blood and to define the underlying mechanism. Freshly isolated CLL cells from 35 patients, as well as normal lymphocytes and platelets, were incubated with various microtubule-disrupting agents plus ABT-737 to assess sensitivity to the single agents and the combination. ABT-737 and vinblastine displayed a range of sensitivity as single agents, and vinblastine markedly sensitized all CLL samples to ABT-737 within six hours. Vinblastine potently induced the proapoptotic protein PMAIP1 (NOXA) in both time- and dose-dependent manner and this was required for the observed apoptosis. Combretastatin A4, which dissociates microtubules by binding to a different site, had the same effect, confirming that interaction of these agents with microtubules is the initial target. Similarly, vincristine and vinorelbine induced NOXA and enhanced CLL sensitivity to ABT-737. Furthermore, vinblastine plus ABT-737 overcame stroma-mediated resistance to ABT-737 alone. Apoptosis was induced with clinically achievable concentrations with no additional toxicity to normal lymphocytes or platelets. These results suggest that vinca alkaloids may improve the clinical efficacy of ABT-263 in patients with CLL. Mol Cancer Ther; 12(8); 1504–14. ©2013 AACR.

Gossypol overcomes stroma-mediated resistance to the BCL2 inhibitor ABT-737 in chronic lymphocytic leukemia cells ex vivo

Gossypol overcomes stroma-mediated resistance to the BCL2 inhibitor ABT-737 in chronic lymphocytic leukemia cells ex vivo

Vinblastine sensitizes leukemia cells to cyclin-dependent kinase inhibitors, inducing acute cell cycle phase-independent apoptosis

The efficacy of many chemotherapeutic agents can be attenuated by expression of the anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1. Flavopiridol and dinaciclib are cyclin-dependent kinase 7 and 9 inhibitors that transcriptionally inhibit expression of Mcl-1. We have investigated the ability of flavopiridol and dinaciclib to sensitize a panel of leukemia cell lines to vinblastine and paclitaxel. Both drugs acutely sensitized most of the leukemia lines to vinblastine, with 100% apoptosis in 4 h. Furthermore, dinaciclib sensitized freshly isolated chronic lymphocytic leukemia cells to vinblastine. This rapid induction of apoptosis was attributed to vinblastine-mediated activation of JNK because (a) flavopiridol and dinaciclib failed to induce apoptosis when combined with non-JNK activating concentrations of vinblastine; (b) JNK inhibitors suppressed JNK activity and prevented apoptosis; (c) flavopiridol did not potentiate apoptosis induced by paclitaxel which does not activate JNK in these cells; and (d) Jurkat cells failed to activate JNK in response to vinblastine and were not sensitive to combinations of vinblastine and flavopiridol or dinaciclib. The rapid induction of apoptosis by this combination in multiple cell systems but not in normal lymphocytes provides justification for performing a clinical trial to assess the efficacy in patients.

Strategically Timing Inhibition of Phosphatidylinositol 3-Kinase to Maximize Therapeutic Index in Estrogen Receptor Alpha–Positive, PIK3CA-Mutant Breast Cancer

Purpose: Phosphatidylinositol 3-kinase (PI3K) inhibitors are being developed for the treatment of estrogen receptor α (ER)–positive breast cancer in combination with antiestrogens. Understanding the temporal response and pharmacodynamic effects of PI3K inhibition in ER+ breast cancer will provide a rationale for treatment scheduling to maximize therapeutic index. Experimental Design: Antiestrogen-sensitive and antiestrogen-resistant ER+ human breast cancer cell lines and mice bearing PIK3CA-mutant xenografts were treated with the antiestrogen fulvestrant, the PI3K inhibitor GDC-0941 (pictilisib; varied doses/schedules that provided similar amounts of drug each week), or combinations. Cell viability, signaling pathway inhibition, proliferation, apoptosis, tumor volume, and GDC-0941 concentrations in plasma and tumors were temporally measured. Results: Treatment with the combination of fulvestrant and GDC-0941, regardless of dose/schedule, was significantly more effective than that with single-agent treatments in fulvestrant-resistant tumors. Short-term, complete PI3K inhibition blocked cell growth in vitro more effectively than chronic, incomplete inhibition. Longer-term PI3K inhibition hypersensitized cells to growth factor signaling upon drug withdrawal. Different schedules of GDC-0941 elicited similar tumor responses. While weekly high-dose GDC-0941 with fulvestrant continuously suppressed PI3K signaling for 72 hours, inducing a bolus of apoptosis and inhibiting proliferation, PI3K reactivation upon GDC-0941 washout induced a proliferative burst. Fulvestrant with daily low-dose GDC-0941 metronomically suppressed PI3K for 6 to 9 hours/day, repeatedly inducing small amounts of apoptosis and temporarily inhibiting proliferation, followed by proliferative rebound compared with fulvestrant alone. Conclusions: Continuous and metronomic PI3K inhibition elicits robust anticancer effects in ER+, PIK3CA-mutant breast cancer. Clinical exploration of alternate treatment schedules of PI3K inhibitors with antiestrogens is warranted. Clin Cancer Res; 22(9); 2250–60. ©2016 AACR. See related commentary by Toska and Baselga, p. 2099

Toward a cure for chronic lymphocytic leukemia: an attack on multiple fronts

Recent developments in molecular therapeutics have armed us with powerful tools to mount a multifaceted offensive against CLL, of which BCL2-targeting agents are a critical element. A strategy consisting of initial debulking of leukemia cells in the periphery (chemoimmunotherapy), fostering exit of neoplastic B cells from the protective environment (B-cell receptor-targeting agents) and killing cells in the protective niche (drug combinations) is poised to dramatically reduce residual disease, prolong progression-free survival, increase overall survival and ultimately improve the lives of millions of CLL patients. We believe it is time to start using all these drugs in rationally designed combination trials.

Manipulating the apoptotic pathway: potential therapeutics for cancer patients

This review summarizes the current state of scientific understanding of the apoptosis pathway, with a focus on the proteins involved in the pathway, their interactions and functions. This forms the rationale for detailing the preclinical and clinical pharmacology of drugs that modulate the pivotal proteins in this pathway, with emphasis on drugs that are furthest advanced in clinical development as anticancer agents. There is a focus on describing drugs that modulate three of the most promising targets in the apoptosis pathway, namely antibodies that bind and activate the death receptors, small molecules that inhibit the anti-apoptotic Bcl-2 family proteins, and small molecules and antisense oligonucleotides that inactivate the inhibitors of apoptosis, all of which drive the equilibrium of the apoptotic pathway towards apoptosis. These structurally different yet functionally related groups of drugs represent a promising novel approach to anticancer therapeutics whether used as monotherapy or in combination with either classical cytotoxic or other molecularly targeted anticancer agents.

Rapid induction of apoptosis in chronic lymphocytic leukemia cells by the microtubule disrupting agent BNC105

ABSTRACT Microtubule targeting agents, such as vinblastine, are usually thought to arrest cells in mitosis and subsequently induce apoptosis. However, they can also cause rapid induction of apoptosis in a cell-cycle phase independent manner. BNC105 is a novel vascular and microtubule disrupting drug that also induces apoptosis rapidly but with markedly increased potency compared to vinca alkaloids and combretastatin A4. BNC105 binds to the colchicine-binding site on tubulin resulting in activation of c-Jun N-terminal kinase (JNK), phosphorylation of ATF2, and induction of ATF3 and Noxa leading to acute apoptosis in chronic lymphocytic leukemia (CLL) cells. Apoptosis induced by BNC105 is dependent upon both JNK activation and Noxa induction. Normal leukocytes and one CLL sample also exhibited JNK activation but not Noxa induction and were resistant to BNC105. This study emphasizes the importance of Noxa and JNK for induction of apoptosis in CLL cells by microtubule targeting drugs, and highlights the potential of BNC105 as a potent therapeutic to treat haematopoietic malignancies.