BH3 mimetic drugs cooperate with Temozolomide, JQ1 and inducers of ferroptosis in killing glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer, with treatment options often constrained due to inherent resistance of malignant cells to conventional therapy. We investigated the impact of triggering programmed cell death (PCD) by using BH3 mimetic drugs in human GBM cell lines. We demonstrate that co-targeting the pro-survival proteins BCL-XL and MCL-1 was more potent at killing six GBM cell lines compared to conventional therapy with Temozolomide or the bromodomain inhibitor JQ1 in vitro. Enhanced cell killing was observed in U251 and SNB-19 cells in response to dual treatment with TMZ or JQ1 combined with a BCL-XL inhibitor, compared to single agent treatment. This was reflected in abundant cleavage/activation of caspase-3 and cleavage of PARP1, markers of apoptosis. U251 and SNB-19 cells were more readily killed by a combination of BH3 mimetics targeting BCL-XL and MCL-1 as opposed to dual treatment with the BCL-2 inhibitor Venetoclax and a BCL-XL inhibitor. The combined loss of BAX and BAK, the essential executioners of intrinsic apoptosis, rendered U251 and SNB-19 cells refractory to any of the drug combinations tested, demonstrating that apoptosis is responsible for their killing. In an orthotopic mouse model of GBM, we demonstrate that the BCL-XL inhibitor A1331852 can penetrate the brain, with A1331852 detected in both tumour and healthy brain regions. We also investigated the impact of combining small molecule inducers of ferroptosis, erastin and RSL3, with BH3 mimetic drugs. We found that a BCL-XL or an MCL-1 inhibitor potently cooperates with inducers of ferroptosis in killing U251 cells. Overall, these findings demonstrate the potential of dual targeting of distinct PCD signalling pathways in GBM and may guide the utility of BCL-XL inhibitors and inducers of ferroptosis with standard of care treatment for improved therapies for GBM.

BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.

Defining the susceptibility of colorectal cancers to BH3-mimetic compounds.

Novel targets are required to improve the outcomes for patients with colorectal cancers. In this regard, the selective inhibitor of the pro-survival protein BCL2, venetoclax, has proven highly effective in several hematological malignancies. In addition to BCL2, potent and highly selective small molecule inhibitors of its relatives, BCLxL and MCL1, are now available, prompting us to investigate the susceptibility of colorectal cancers to the inhibition of one or more of these pro-survival proteins. While targeting BCLxL, but not BCL2 or MCL1, on its own had some impact, most (15/17) of the immortalized colorectal cancer cell lines studied were efficiently killed by the combined targeting of BCLxL and MCL1. Importantly, these in vitro findings were confirmed in a xenograft model and, interestingly, in all (5/5) patient derived tumor organoids evaluated. Our results lend strong support to the notion that BCLxL and MCL1 are highly promising targets for further evaluation in efforts to improve the treatment of colorectal cancers.

Simplified silvestrol analogues with potent cytotoxic activity.

The complex natural products silvestrol (1) and episilvestrol (2) are inhibitors of translation initiation through binding to the DEAD-box helicase eukaryotic initiation factor 4A (eIF4A). Both compounds are potently cytotoxic to cancer cells in vitro, and 1 has demonstrated efficacy in vivo in several xenograft cancer models. Here we show that 2 has limited plasma membrane permeability and is metabolized in liver microsomes in a manner consistent with that reported for 1. In addition, we have prepared a series of analogues of these compounds where the complex pseudo-sugar at C6 has been replaced with chemically simpler moieties to improve drug-likeness. Selected compounds from this work possess excellent activity in biochemical and cellular translation assays with potent activity against leukemia cell lines.

Crystallization and preliminary X-ray characterization of Epstein-Barr virus BHRF1 in complex with a benzoylurea peptidomimetic.

BHRF1 is a pro-survival Bcl-2 homologue encoded by Epstein-Barr virus (EBV) that plays a key role in preventing premature host cell death during viral infection and may contribute to the development of malignancies associated with chronic EBV infections. The anti-apoptotic action of BHRF1 is based on its ability to sequester pro-apoptotic Bcl-2 family proteins, in particular Bim and Bak. These interactions have been previously studied in three dimensions by determining crystal structures of BHRF1 in complex with both Bim and Bak BH3 domains. Screening of a library of peptidomimetic compounds based on the benzoylurea scaffold that mimics critical Bim BH3 domain side chains against BHRF1 led to the identification of an inhibitor of BHRF1 that displays micromolar affinity. Single crystals were obtained from the co-crystallization of recombinant BHRF1 protein with this peptidomimetic compound. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=66.8, b=91.1, c=151.9 Å. Diffraction data were collected to 2.11 Šresolution on the MX2 beamline at the Australian Synchrotron.