In situ visualization of endothelial cell-derived extracellular vesicle formation in steady state and malignant conditions.

Endothelial cells are integral components of all vasculature within complex organisms. As they line the blood vessel wall, endothelial cells are constantly exposed to a variety of molecular factors and shear force that can induce cellular damage and stress. However, how endothelial cells are removed or eliminate unwanted cellular contents, remains unclear. The generation of large extracellular vesicles (EVs) has emerged as a key mechanism for the removal of cellular waste from cells that are dying or stressed. Here, we used intravital microscopy of the bone marrow to directly measure the kinetics of EV formation from endothelial cells in vivo under homoeostatic and malignant conditions. These large EVs are mitochondria-rich, expose the 'eat me' signal phosphatidylserine, and can interact with immune cell populations as a potential clearance mechanism. Elevated levels of circulating EVs correlates with degradation of the bone marrow vasculature caused by acute myeloid leukaemia. Together, our study provides in vivo spatio-temporal characterization of EV formation in the murine vasculature and suggests that circulating, large endothelial cell-derived EVs can provide a snapshot of vascular damage at distal sites.

Induction of endoplasmic reticulum stress is associated with the anti-tumor activity of monepantel across cancer types.

BACKGROUND: Monepantel is an anti-helminthic drug that also has anti-cancer properties. Despite several studies over the years, the molecular target of monepantel in mammalian cells is still unknown, and its mechanism-of-action is not fully understood, though effects on cell cycle, mTOR signalling and autophagy have been implicated. METHODS: Viability assays were performed on >20 solid cancer cell cells, and apoptosis assays were performed on a subset of these, including 3D cultures. Genetic deletion of BAX/BAK and ATG were used to establish roles of apoptosis and autophagy in killing activity. RNA-sequencing was performed on four cell lines after monepantel treatment, and differentially regulated genes were confirmed by Western blotting. RESULTS: We showed that monepantel has anti-proliferative activity on a broad range of cancer cell lines. In some, this was associated with induction of apoptosis which was confirmed using a BAX/BAK-deficient cell line. However, proliferation is still inhibited in these cells following monepantel treatment, indicating cell-cycle disruption as the major anti-cancer effect. Previous studies have also indicated autophagic cell death occurs following monepantel treatment. We showed autophagy induction in multiple cell lines; however, deletion of a key autophagy regulator ATG7 had minimal impact on monepantel's anti-proliferative activity, suggesting autophagy is associated with, but not required for its anti-tumour effects. Transcriptomic analysis of four cell lines treated with monepantel revealed downregulation of many genes involved in the cell cycle, and upregulation of genes linked to ATF4-mediated ER stress responses, especially those involved in amino-acid metabolism and protein synthesis. CONCLUSIONS: As these outcomes are all associated with mTOR signalling, cell cycle and autophagy, we now provide a likely triggering mechanism for the anti-cancer activity of monepantel.

A novel BH3-mimetic, AZD0466, targeting BCL-XL and BCL-2 is effective in pre-clinical models of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with treatment limited to Cisplatin and Pemetrexed chemotherapy. Recently, we showed that drugs targeting the BCL-2-regulated apoptosis pathway could kill MPM cell lines in vitro, and control tumor growth in vivo. These studies showed BCL-XL was the dominant pro-survival BCL-2 family member correlating with its high-level expression in cells and patient tumor samples. In this study we show another inhibitor, AZD4320 that targets BCL-XL (and BCL-2), can also potently kill MPM tumor cells in vitro (EC50 values in the 200 nM range) and this effect is enhanced by co-inhibition of MCL-1 using AZD5991. Moreover, we show that a novel nanoparticle, AZD0466, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer, was as effective as standard-of-care chemotherapy, Cisplatin, at inhibiting tumor growth in mouse xenograft studies, and this effect was enhanced when both drugs were combined. Critically, the degree of thrombocytopenia, an on-target toxicity associated with BCL-XL inhibition, was significantly reduced throughout the treatment period compared to other BCL-XL-targeting BH3-mimetics. These pre-clinical findings provide a rationale for the future clinical evaluation for novel BH3-mimetic formulations in MPM, and indeed, other solid tumor types dependent on BCL-XL.

ATF3 Repression of BCL-XL Determines Apoptotic Sensitivity to HDAC Inhibitors across Tumor Types.

Purpose: Histone deacetylase inhibitors (HDACi) are epigenome-targeting small molecules approved for the treatment of cutaneous T-cell lymphoma and multiple myeloma. They have also demonstrated clinical activity in acute myelogenous leukemia, non-small cell lung cancer, and estrogen receptor-positive breast cancer, and trials are underway assessing their activity in combination regimens including immunotherapy. However, there is currently no clear strategy to reliably predict HDACi sensitivity. In colon cancer cells, apoptotic sensitivity to HDACi is associated with transcriptional induction of multiple immediate-early (IE) genes. Here, we examined whether this transcriptional response predicts HDACi sensitivity across tumor type and investigated the mechanism by which it triggers apoptosis.Experimental Design: Fifty cancer cell lines from diverse tumor types were screened to establish the correlation between apoptotic sensitivity, induction of IE genes, and components of the intrinsic apoptotic pathway.Results: We show that sensitivity to HDACi across tumor types is predicted by induction of the IE genes FOS, JUN, and ATF3, but that only ATF3 is required for HDACi-induced apoptosis. We further demonstrate that the proapoptotic function of ATF3 is mediated through direct transcriptional repression of the prosurvival factor BCL-XL (BCL2L1) These findings provided the rationale for dual inhibition of HDAC and BCL-XL, which we show strongly cooperate to overcome inherent resistance to HDACi across diverse tumor cell types.Conclusions: These findings explain the heterogeneous responses of tumor cells to HDACi-induced apoptosis and suggest a framework for predicting response and expanding their therapeutic use in multiple cancer types. Clin Cancer Res; 23(18); 5573-84. ©2017 AACR.

MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib.

BACKGROUND: Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. METHODS: To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. RESULTS: MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. CONCLUSION: These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.

Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM.

A large proportion of melanomas harbour the activating BRAFV600E mutation that renders these cells dependent on MAPK signalling for their survival. Although the highly specific and clinically approved BRAFV600E kinase inhibitor, PLX4032, induces apoptosis of melanoma cells bearing this mutation, the underlying molecular mechanisms are not fully understood. Here, we reveal that PLX4032-induced apoptosis depends on the induction of the pro-apoptotic BH3-only protein PUMA with a minor contribution of its relative BIM. Apoptosis could be significantly augmented when PLX4032 was combined with an inhibitor of the pro-survival protein BCL-XL, whereas neutralization of the pro-survival family member BCL-2 caused no additional cell death. Although the initial response to PLX4032 in melanoma patients is very potent, resistance to the drug eventually develops and relapse occurs. Several factors can cause melanoma cells to develop resistance to PLX4032; one of them is the activation of the receptor tyrosine kinase cMET on melanoma cells by its ligand, hepatocyte growth factor (HGF), provided by the tumour microenvironment or the cancer cells themselves. We found that HGF mediates resistance of cMET-expressing BRAF mutant melanoma cells to PLX4032-induced apoptosis through downregulation of PUMA and BIM rather than by increasing the expression of pro-survival BCL-2-like proteins. These results suggest that resistance to PLX4032 may be overcome by specifically increasing the levels of PUMA and BIM in melanoma cells through alternative signalling cascades or by blocking pro-survival BCL-2 family members with suitable BH3 mimetic compounds.

Bcl-2, Bcl-x(L), and Bcl-w are not equivalent targets of ABT-737 and navitoclax (ABT-263) in lymphoid and leukemic cells.

The BH3-mimetic ABT-737 and an orally bioavailable compound of the same class, navitoclax (ABT-263), have shown promising antitumor efficacy in preclinical and early clinical studies. Although both drugs avidly bind Bcl-2, Bcl-x(L), and Bcl-w in vitro, we find that Bcl-2 is the critical target in vivo, suggesting that patients with tumors overexpressing Bcl-2 will probably benefit. In human non-Hodgkin lymphomas, high expression of Bcl-2 but not Bcl-x(L) predicted sensitivity to ABT-263. Moreover, we show that increasing Bcl-2 sensitized normal and transformed lymphoid cells to ABT-737 by elevating proapoptotic Bim. In striking contrast, increasing Bcl-x(L) or Bcl-w conferred robust resistance to ABT-737, despite also increasing Bim. Cell-based protein redistribution assays unexpectedly revealed that ABT-737 disrupts Bcl-2/Bim complexes more readily than Bcl-x(L)/Bim or Bcl-w/Bim complexes. These results have profound implications for how BH3-mimetics induce apoptosis and how the use of these compounds can be optimized for treating lymphoid malignancies.

The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins.

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim's proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.