Bruton's tyrosine kinase inhibition increases BCL-2 dependence and enhances sensitivity to venetoclax in chronic lymphocytic leukemia.

Although the BTK inhibitor ibrutinib has transformed the management of patients with chronic lymphocytic leukemia (CLL), it does not induce substantial apoptosis in vitro, and as such the mechanisms underlying its ability to kill CLL cells are not well understood. Acalabrutinib, a more specific BTK inhibitor now in development, also appears to be highly effective in CLL, but the connection of its mechanism with CLL cell death is also unclear. Using dynamic BH3 profiling, we analyzed alterations in the function of the mitochondrial apoptotic pathway induced by ibrutinib and acalabrutinib. We studied CLL patient samples treated ex vivo with both drugs, as well as primary samples from CLL patients on clinical trials of both drugs. We found that BTK inhibition enhances mitochondrial BCL-2 dependence without significantly altering overall mitochondrial priming. Enhancement of BCL-2 dependence was accompanied by an increase in the pro-apoptotic protein BIM. In contrast, treatment with the selective BCL-2 inhibitor venetoclax enhanced overall mitochondrial priming without increasing BCL-2 dependence. Pre-treatment of CLL cells with either BTK inhibitor, whether ex vivo or in vivo in patients, enhanced killing by venetoclax. Our data suggest that BTK inhibition enhances mitochondrial BCL-2 dependence, supporting the ongoing development of clinical trials combining BTK and BCL-2 inhibition.

Constitutive p53 heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors of BCL-xL.

Proapoptotic molecules directly targeting the BCL-2 family network are promising anticancer therapeutics, but an understanding of the cellular stress signals that render them effective is still elusive. We show here that the tumor suppressor p53, at least in part by transcription independent mechanisms, contributes to cell death induction and full activation of BAX by BH3 mimetic inhibitors of BCL-xL. In addition to mildly facilitating the ability of compounds to derepress BAX from BCL-xL, p53 also provides a death signal downstream of anti-apoptotic proteins inhibition. This death signal cooperates with BH3-induced activation of BAX and it is independent from PUMA, as enhanced p53 can substitute for PUMA to promote BAX activation in response to BH3 mimetics. The acute sensitivity of mitochondrial priming to p53 revealed here is likely to be critical for the clinical use of BH3 mimetics.

Activity of a selective inhibitor of nuclear export, selinexor (KPT-330), against AML-initiating cells engrafted into immunosuppressed NSG mice.

Currently available combination chemotherapy for acute myeloid leukemia (AML) often fails to result in long-term remissions, emphasizing the need for novel therapeutic strategies. We reasoned that targeted inhibition of a prominent nuclear exporter, XPO1/CRM1, could eradicate self-renewing leukemia-initiating cells (LICs) whose survival depends on timely XPO1-mediated transport of specific protein and RNA cargoes. Using an immunosuppressed mouse model bearing primary patient-derived AML cells, we demonstrate that selinexor (KPT-330), an oral antagonist of XPO1 that is currently in clinical trials, has strong activity against primary AML cells while sparing normal stem and progenitor cells. Importantly, limiting dilution transplantation assays showed that this cytotoxic activity is not limited to the rapidly proliferating bulk population of leukemic cells but extends to the LICs, whose inherent drug resistance and unrestricted self-renewal capacity has been implicated in the difficulty of curing AML patients with conventional chemotherapy alone.

Bcl-xL controls a switch between cell death modes during mitotic arrest.

Antimitotic agents such as microtubule inhibitors (paclitaxel) are widely used in cancer therapy while new agents blocking mitosis onset are currently in development. All these agents impose a prolonged mitotic arrest in cancer cells that relies on sustained activation of the spindle assembly checkpoint and may lead to subsequent cell death by incompletely understood molecular events. We have investigated the role played by anti-apoptotic Bcl-2 family members in the fate of mitotically arrested mammary tumor cells treated with paclitaxel, or depleted in Cdc20, the activator of the anaphase promoting complex. Under these conditions, a weak and delayed mitotic cell death occurs that is caspase- and Bax/Bak-independent. Moreover, BH3 profiling assays indicate that viable cells during mitotic arrest are primed to die by apoptosis and that Bcl-xL is required to maintain mitochondrial integrity. Consistently, Bcl-xL depletion, or treatment with its inhibitor ABT-737 (but not with the specific Bcl-2 inhibitor ABT-199), during mitotic arrest converts cell response to antimitotics to efficient caspase and Bax-dependent apoptosis. Apoptotic priming under conditions of mitotic arrest relies, at least in part, on the phosphorylation on serine 62 of Bcl-xL, which modulates its interaction with Bax and its sensitivity to ABT-737. The phospho-mimetic S62D-Bcl-xL mutant is indeed less efficient than the corresponding phospho-deficient S62A-Bcl-xL mutant in sequestrating Bax and in protecting cancer cells from mitotic cell death or yeast cells from Bax-induced growth inhibition. Our results provide a rationale for combining Bcl-xL targeting to antimitotic agents to improve clinical efficacy of antimitotic strategy in cancer therapy.

Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells.

Tumor heterogeneity is in part determined by the existence of cancer stem cells (CSCs) and more differentiated tumor cells. CSCs are considered to be the tumorigenic root of cancers and suggested to be chemotherapy resistant. Here we exploited an assay that allowed us to measure chemotherapy-induced cell death in CSCs and differentiated tumor cells simultaneously. This confirmed that CSCs are selectively resistant to conventional chemotherapy, which we revealed is determined by decreased mitochondrial priming. In agreement, lowering the anti-apoptotic threshold using ABT-737 and WEHI-539 was sufficient to enhance chemotherapy efficacy, whereas ABT-199 failed to sensitize CSCs. Our data therefore point to a crucial role of BCLXL in protecting CSCs from chemotherapy and suggest that BH3 mimetics, in combination with chemotherapy, can be an efficient way to target chemotherapy-resistant CSCs.

Modulation of Mcl-1 sensitizes glioblastoma to TRAIL-induced apoptosis.

Glioblastoma (GBM) is the most aggressive form of primary brain tumour, with dismal patient outcome. Treatment failure is associated with intrinsic or acquired apoptosis resistance and the presence of a highly tumourigenic subpopulation of cancer cells called GBM stem cells. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising novel therapy for some treatment-resistant tumours but unfortunately GBM can be completely resistant to TRAIL monotherapy. In this study, we identified Mcl-1, an anti-apoptotic Bcl-2 family member, as a critical player involved in determining the sensitivity of GBM to TRAIL-induced apoptosis. Effective targeting of Mcl-1 in TRAIL resistant GBM cells, either by gene silencing technology or by treatment with R-roscovitine, a cyclin-dependent kinase inhibitor that targets Mcl-1, was demonstrated to augment sensitivity to TRAIL, both within GBM cells grown as monolayers and in a 3D tumour model. Finally, we highlight that two separate pathways are activated during the apoptotic death of GBM cells treated with a combination of TRAIL and R-roscovitine, one which leads to caspase-8 and caspase-3 activation and a second pathway, involving a Mcl-1:Noxa axis. In conclusion, our study demonstrates that R-roscovitine in combination with TRAIL presents a promising novel strategy to trigger cell death pathways in glioblastoma.

p53 regulates a non-apoptotic death induced by ROS.

DNA damage induced by reactive oxygen species and several chemotherapeutic agents promotes both p53 and poly (ADP-ribose) polymerase (PARP) activation. p53 activation is well known to regulate apoptotic cell death, whereas robust activation of PARP-1 has been shown to promote a necrotic cell death associated with energetic collapse. Here we identify a novel role for p53 in modulating PARP enzymatic activity to regulate necrotic cell death. In mouse embryonic fibroblasts, human colorectal and human breast cancer cell lines, loss of p53 function promotes resistance to necrotic, PARP-mediated cell death. We therefore demonstrate that p53 can regulate both necrotic and apoptotic cell death, mutations or deletions in this tumor-suppressor protein may be selected by cancer cells to provide not only their resistance to apoptosis but also to necrosis, and explain resistance to chemotherapy and radiation even when it kills via non-apoptotic mechanisms.

The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance.

Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were 'primed for death'. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, ∼70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically.

Displacement of Bim by Bmf and Puma rather than increase in Bim level mediates paclitaxel-induced apoptosis in breast cancer cells.

Taxanes exert their antitumor effect through stabilizing microtubule dynamics and initiating G2/M arrest in cancer cells followed by apoptotic cell death. However, the signaling pathways that connect paclitaxel-induced microtubule perturbation to mitochondrial outer membrane permeabilization and cytochrome c release are not well characterized. Here, we show that in breast cancer cells, paclitaxel induces a novel displacement mechanism: prodeath BH3-only proteins Bmf and Puma competitively displace prodeath BH3-only protein Bim from antiapoptotic proteins to activate Bax and Bak and commit the cell to apoptotic death. Bim and either Puma or Bmf are required for paclitaxel toxicity. Although prior mechanisms of apoptosis induced by taxol have focused on changes in Bim levels, we find that an increase is not required for paclitaxel killing of breast cancer cells. Rather, competitive displacement of Bim from antiapoptotic proteins is the important step committing the cell to death. This novel mechanism suggests the potential usage of novel therapies targeted at altering BH3-only protein heterodimerization.

BIM and other BCL-2 family proteins exhibit cross-species conservation of function between zebrafish and mammals.

Here we investigate the function of zebrafish Bcl-2 family proteins and demonstrate important conservation of function across zebrafish and mammalian systems. We have isolated a zebrafish ortholog of mammalian BIM and show that it is the most toxic of the zebrafish BH3-only genes examined, sharing this characteristic with the mammalian BIM gene. The zebrafish bad gene shows a complete lack of embryonic lethality, but like mammalian BAD, its pro-apoptotic activity is regulated through phosphorylation of critical serines. We also found that the pattern of mitochondrial dysfunction observed by zebrafish BH3 domain peptides in a mammalian cytochrome c release assay recapitulates the pattern of embryonic lethality induced by the respective mRNA injections in vivo. In contrast to zebrafish Bim, Bid exhibited only weak binding to zebrafish Bcl-2 and moderate-to-weak overall lethality in zebrafish embryos and isolated mitochondria. Given that zebrafish Bcl-2 binds strongly to mammalian BID and BIM peptides and proteins, the protein identified as the zebrafish Bid ortholog has different properties than mammalian BID. Overall, our results demonstrate the high degree of functional conservation between zebrafish and mammalian Bcl-2 family proteins, thus validating the zebrafish as a model system to further dissect the molecular mechanisms that regulate apoptosis in future forward genetic and chemical modifier screens.

BH3 domains define selective inhibitory interactions with BHRF-1 and KSHV BCL-2.

The Epstein-Barr and Kaposi's sarcoma gamma-herpesviruses (KSHVs) are associated with certain cancers, and encode B-cell leukemia/lymphoma 2 (BCL-2) homologs, BHRF-1 and KSHV BCL-2, respectively. Little is known, however, about the molecular interactions allowing viral BCL-2 homologs to mediate their anti-apoptotic function. Cellular anti-apoptotic proteins, such as BCL-2 and MCL-1, prevent death via selective interactions with pro-death BH3-only proteins. To investigate whether BHRF-1 and KSHV BCL-2 function similarly, we made recombinant BHRF-1 and KSHV BCL-2 proteins. We identified the individual binding patterns for BHRF-1 and KSHV BCL-2 to BH3 domains. These studies surprisingly showed that KSHV BCL-2 is more closely related to MCL-1 than to BCL-2, a result confirmed by sequence analysis. GST-BHRF-1 and GST-KSHV BCL-2 bound BH3-only family proteins from human cells. BHRF-1 protected mammalian cells from growth factor withdrawal, etoposide and adriamycin. We found that both BCL-2 and BHRF-1 sequestered pro-death BH3-only proteins under growth factor-deficient conditions. Finally, we tested the ability of a panel of BH3 peptides to inhibit BHRF-1 and KSHV BCL-2 function in a mitochondrial model of apoptosis. We found that each could be inhibited by the select group of BH3 peptides identified in our binding assay. Our studies define the biochemical interactions underlying BHRF-1 and KSHV BCL-2 anti-apoptotic function, and identify peptides that are prototypic inhibitors of this function.

Mimicking the BH3 domain to kill cancer cells.

Cancer cells show deviant behavior that induces apoptotic signaling. To survive, cancer cells typically acquire changes enabling evasion of death signals. One way they do this is by increasing the expression of anti-apoptotic BCL-2 proteins. Anti-apoptotic BCL-2 family proteins antagonize death signaling by forming heterodimers with pro-death proteins. Heterodimer formation occurs through binding of the pro-apoptotic protein's BH3 domain into the hydrophobic cleft of anti-apoptotic proteins. The BH3 mimetics are small molecule antagonists of the anti-apoptotic BCL-2 members that function as competitive inhibitors by binding to the hydrophobic cleft. Under certain conditions, antagonism of anti-apoptotic BCL-2 family proteins can unleash pro-death molecules in cancer cells. Thus, the BH3 mimetics are a new class of cancer drugs that specifically target a mechanism of cancer cell survival to selectively kill cancer cells.

BH3 peptidomimetics potently activate apoptosis and demonstrate single agent efficacy in neuroblastoma.

The major impediment to cure for many malignancies is the development of therapy resistance with resultant tumor progression. Genetic alterations leading to subversion of inherent apoptosis pathways are common themes in therapy resistance. Bcl-2 family proteins play a critical role in regulating mitochondrial apoptosis that governs chemotherapeutic effects, and defective engagement of these pathways contributes to treatment failure. We have studied the efficacy of BH3 peptidomimetics consisting of the minimal death, or BH3, domains of the proapoptotic BH3-only proteins Bid and Bad to induce apoptosis using neuroblastoma (NB) as a model system. We demonstrate that BH3 peptides, modified with an arginine homopolymer for membrane transduction (called r8-BidBH3 and r8-BadBH3, respectively), potently induce apoptosis in NB cells, including those with MYCN amplification. Cell death is caspase 9 dependent, consistent with a requirement for the intrinsic mitochondrial pathway. Substitutions at highly conserved residues within the r8-BidBH3 peptide abolish apoptotic efficacy supporting activity through specific BH domain interactions. Concomitant exposure to r8-BadBH3 and r8-BidBH3 at sublethal monotherapy doses revealed potent synergy consistent with a competitive displacement model, whereby BH3 peptides displace sequestered BH3 proteins to induce cell death. Further, BH3 peptides demonstrate antitumor efficacy in a xenograft model of NB in the absence of additional genotoxic or trophic stressors. These data provide proof of principle that targeted re-engagement of apoptosis pathways may be of therapeutic utility, and BH3-like compounds are attractive lead agents to re-establish therapy-induced apoptosis in refractory malignancies.

Cancer, coagulation, and anticoagulation.

Thromboembolic disease affects about 15% of cancer patients and presents a challenge to the oncologist for both prophylaxis and treatment. Although long known to be associated with malignancy, the underlying biochemical mechanisms are poorly understood. Both low-dose warfarin and low molecular weight heparin are effective strategies for prophylaxis of venous thromboembolism, including those involving venous access devices. Current treatment options for venous thromboembolism include heparin (unfractionated and low molecular weight), warfarin, and internal vena cava filters. The appropriate use of these therapeutic options in cancer patients is reviewed herein. There is suggestive evidence that heparin may be superior to warfarin in the long-term treatment of venous thromboembolism. Whether anticoagulants might also improve cancer survival rates independent of their effect on thromboembolism deserves further investigation.

The importance of intramolecular ion pairing in intermediate filaments.

Nuclear and cytoskeletal networks of 10-nm intermediate filaments (IFs) are probably ubiquitous in multicellular eukaryotes. They likely play a role in maintaining the mechanical integrity of a cell. With the exception of the nuclear lamins, IF proteins can form IFs in vitro in the absence of cofactors or associated proteins. Below we present data suggesting that the large alpha-helical "rod" domains of IF proteins are stabilized by large numbers (up to 50) of intra-helical ion pairs formed by residues of opposite charge situated four residues apart. These many ion pairs, sometimes involving up to 30% of the residues within a coiled-coil IF segment, can potentially contribute as much as 10-25 kcal/mol (1 kcal = 4.18 kJ) to the stability of a single alpha-helical rod. Such stabilization is likely to play a major role in the chemical and physical stability of IF networks in vitro and in vivo. An investigation of other coiled-coil proteins shows that selection for intrahelical ion pairing is not simply a property intrinsic to coiled-coil proteins. Rather, there is a correlation between the degree to which there is selection for intrahelical ion pairs and the extent to which a coiled-coil protein participates in highly ordered multimolecular interactions--e.g., as in IFs and myosin thick filaments. The propensity of putative ion pairs in some IF proteins--e.g., epidermal keratins--suggests that an underlying structural stability at the level of the monomer may play an important role in the extraordinary stability of dimers and higher ordered structures in cytoplasmic IFs.

Genetic bases of epidermolysis bullosa simplex and epidermolytic hyperkeratosis.

Keratins are the major structural proteins of the epidermis. Analyzing keratin gene sequences, appreciating the switch in keratin gene expression that takes place as epidermal cells commit to terminally differentiate, and elucidating how keratins assemble into 10-nm filaments have provided the foundation that has led to the discoveries of the genetic bases of two major classes of human skin diseases. In this report, we review the cell biology and human genetics of these diseases, epidermolysis bullosa simplex and epidermolytic hyperkeratosis. Both of these diseases are epidermal disorders of keratin, typified by cell fragility as a consequence of defects in the mechanical strength of basal epidermolysis bullosa simplex or suprabasal epidermolytic hyperkeratosis cells.

Disease severity correlates with position of keratin point mutations in patients with epidermolysis bullosa simplex.

Keratins are the major structural proteins of the epidermis. Recently, it was discovered that point mutations in the epidermal keratins can lead to the blistering skin diseases epidermolysis bullosa simplex (EBS) and epidermolytic hyperkeratosis (EH), involving epidermal cell fragility and rupture upon mechanical stress. In this study, we demonstrate a correlation between disease severity, location of point mutations within the keratin polypeptides, and degree to which these mutations perturb keratin filament structure. Interestingly, of the 11 EBS or EH mutations thus far identified, 6 affect a single highly evolutionarily conserved arginine residue, which, when mutated, markedly perturbs keratin filament structure and keratin network formation. This site also appears to be a hot spot for mutation by CpG methylation and deamination. In the four epidermal keratins, there are several other CpG dinucleotides that exist at codons within the highly conserved ends of the keratin rod. To elucidate why mutations at these sites have not been detected in severe cases of EBS, we engineered 7 of these C-->T transitions in K14 and tested their ability to perturb keratin network formation and keratin filament assembly in vitro. The effects of these mutants on keratin filament network formation were significantly less severe than the EBS/EH arginine mutation, suggesting that the high incidence of mutations of the residue in EBS and EH patients is a result of both a special sensitivity of filament structure to perturbations in this residue and its susceptibility to mutagenesis.