Patrick Bhola

Mitochondria—Judges and Executioners of Cell Death Sentences

Apoptosis is a form of programmed cell death that is critical for basic human development and physiology. One of the more important surprises in cell biology in the last two decades is the extent to which mitochondria represent a physical point of convergence for many apoptosis-inducing signals in mammalian cells. Mitochondria not only adjudicate the decision of whether or not to commit to cell death, but also release toxic proteins culminating in widespread proteolysis, nucleolysis, and cell engulfment. Interactions among BCL-2 family proteins at the mitochondrial outer membrane control the release of these toxic proteins and, by extension, control cellular commitment to apoptosis. This pathway is particularly relevant to cancer treatment, as most cancer chemotherapies trigger mitochondrial-mediated apoptosis. In this Review, we discuss recent advances in the BCL-2 family interactions, their control by upstream factors, and how the mitochondria itself alters these interactions. We also highlight recent clinical insights into mitochondrial-mediated apoptosis and novel cancer therapies that exploit this pathway.

Sensitivity analysis of intracellular signaling pathway kinetics predicts targets for stem cell fate control.

Directing stem cell fate requires knowledge of how signaling networks integrate temporally and spatially segregated stimuli. We developed and validated a computational model of signal transducer and activator of transcription-3 (Stat3) pathway kinetics, a signaling network involved in embryonic stem cell (ESC) self-renewal. Our analysis identified novel pathway responses; for example, overexpression of the receptor glycoprotein-130 results in reduced pathway activation and increased ESC differentiation. We used a systematic in silico screen to identify novel targets and protein interactions involved in Stat3 activation. Our analysis demonstrates that signaling activation and desensitization (the inability to respond to ligand restimulation) is regulated by balancing the activation state of a distributed set of parameters including nuclear export of Stat3, nuclear phosphatase activity, inhibition by suppressor of cytokine signaling, and receptor trafficking. This knowledge was used to devise a temporally modulated ligand delivery strategy that maximizes signaling activation and leads to enhanced ESC self-renewal.

Developmental Regulation of Mitochondrial Apoptosis by c-Myc Governs Age- and Tissue-Specific Sensitivity to Cancer Therapeutics.

It is not understood why healthy tissues can exhibit varying levels of sensitivity to the same toxic stimuli. Using BH3 profiling, we find that mitochondria of many adult somatic tissues, including brain, heart, and kidneys, are profoundly refractory to pro-apoptotic signaling, leading to cellular resistance to cytotoxic chemotherapies and ionizing radiation. In contrast, mitochondria from these tissues in young mice and humans are primed for apoptosis, predisposing them to undergo cell death in response to genotoxic damage. While expression of the apoptotic protein machinery is nearly absent by adulthood, in young tissues its expression is driven by c-Myc, linking developmental growth to cell death. These differences may explain why pediatric cancer patients have a higher risk of developing treatment-associated toxicities.

Determinism and divergence of apoptosis susceptibility in mammalian cells.

Although the cellular decision to commit to apoptosis is important for organism homeostasis, there is considerable variability in the onset of apoptosis between cells, even in clonal populations. Using live single-cell imaging, we observed that the onset of apoptotic proteolytic activity was tightly synchronized between nearby cells. This synchrony was not a consequence of secreted factors and was not correlated to the cell cycle. The synchrony was only seen amongst related cells and was lost over successive generations. The times of apoptosis also diverged within a generation, but this was blocked by inhibiting protein synthesis before triggering apoptosis. These results suggest that the cell-cell variability of apoptosis times is due to the divergence of the molecular composition of the cell, and that the decision to commit to apoptosis at the time of drug addition is a deterministic decision.

Targeted apoptosis of myofibroblasts with the BH3 mimetic ABT-263 reverses established fibrosis

ABT-263 blocks BCL-XL to induce apoptosis in human dermal fibroblasts from patients with scleroderma and in myofibroblasts in mice, reversing dermal fibrosis. Reversing fibrosis through apoptosis Myofibroblasts are integral in a feedback loop that perpetuates fibrosis through stiffening of the extracellular matrix. Lagares et al. determined that proapoptotic proteins are increased in these stiffness-activated myofibroblasts, and these cells become dependent on antiapoptotic protein expression to prevent their death. A drug that mimics a proapoptotic protein to block the antiapoptotic protein BCL-XL induced apoptosis in fibroblasts from patients with scleroderma, a fibrotic connective tissue disorder. The drug, ABT-263, was also effective in reversing established fibrosis in a mouse model of scleroderma. This study suggests that targeting antiapoptotic proteins to induce myofibroblast apoptosis could be an effective strategy to treat fibrosis. Persistent myofibroblast activation distinguishes pathological fibrosis from physiological wound healing, suggesting that therapies selectively inducing myofibroblast apoptosis could prevent progression and potentially reverse established fibrosis in diseases such as scleroderma, a heterogeneous autoimmune disease characterized by multiorgan fibrosis. We demonstrate that fibroblast-to-myofibroblast differentiation driven by matrix stiffness increases the mitochondrial priming (proximity to the apoptotic threshold) of these activated cells. Mitochondria in activated myofibroblasts, but not quiescent fibroblasts, are primed by death signals such as the proapoptotic BH3-only protein BIM, which creates a requirement for tonic expression of the antiapoptotic protein BCL-XL to sequester BIM and ensure myofibroblast survival. Myofibroblasts become particularly susceptible to apoptosis induced by “BH3 mimetic” drugs inhibiting BCL-XL such as ABT-263. ABT-263 displaces BCL-XL binding to BIM, allowing BIM to activate apoptosis on stiffness-primed myofibroblasts. Therapeutic blockade of BCL-XL with ABT-263 (navitoclax) effectively treats established fibrosis in a mouse model of scleroderma dermal fibrosis by inducing myofibroblast apoptosis. Using a BH3 profiling assay to assess mitochondrial priming in dermal fibroblasts derived from patients with scleroderma, we demonstrate that the extent of apoptosis induced by BH3 mimetic drugs correlates with the extent of their mitochondrial priming, indicating that BH3 profiling could predict apoptotic responses of fibroblasts to BH3 mimetic drugs in patients with scleroderma. Together, our findings elucidate the potential efficacy of targeting myofibroblast antiapoptotic proteins with BH3 mimetic drugs in scleroderma and other fibrotic diseases.

MEF2C phosphorylation is required for chemotherapy resistance in acute myeloid leukemia

In acute myeloid leukemia (AML), chemotherapy resistance remains prevalent and poorly understood. Using functional proteomics of patient AML specimens, we identified MEF2C S222 phosphorylation as a specific marker of primary chemoresistance. We found that Mef2cS222A/S222A knock-in mutant mice engineered to block MEF2C phosphorylation exhibited normal hematopoiesis, but were resistant to leukemogenesis induced by MLL-AF9 MEF2C phosphorylation was required for leukemia stem cell maintenance and induced by MARK kinases in cells. Treatment with the selective MARK/SIK inhibitor MRT199665 caused apoptosis and conferred chemosensitivity in MEF2C-activated human AML cell lines and primary patient specimens, but not those lacking MEF2C phosphorylation. These findings identify kinase-dependent dysregulation of transcription factor control as a determinant of therapy response in AML, with immediate potential for improved diagnosis and therapy for this disease.Significance: Functional proteomics identifies phosphorylation of MEF2C in the majority of primary chemotherapy-resistant AML. Kinase-dependent dysregulation of this transcription factor confers susceptibility to MARK/SIK kinase inhibition in preclinical models, substantiating its clinical investigation for improved diagnosis and therapy of AML. Cancer Discov; 8(4); 478-97. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.

Synergistic interactions with PI3K inhibition that induce apoptosis

Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a PIK3CA mutant human breast cancer cell. We identified 5 genes (PIM2, ZAK, TACC1, ZFR, ZNF565) whose suppression induced cell death upon PI3K inhibition. We showed that small molecule inhibitors of the PIM2 and ZAK kinases synergize with PI3K inhibition. In addition, using a microscale implementable device to deliver either siRNAs or small molecule inhibitors in vivo, we showed that suppressing these 5 genes with PI3K inhibition induced tumor regression. These observations identify targets whose inhibition synergizes with PI3K inhibitors and nominate potential combination therapies involving PI3K inhibition. DOI: http://dx.doi.org/10.7554/eLife.24523.001

Abstract 4728: Apoptotic priming is regulated by a developmental program and predisposes children to therapy-induced toxicity

Pediatric cancer patients frequently suffer higher levels of treatment-induced toxicities than adults, limiting the use of potentially curative therapies. For example, brain irradiation contributes to the cure of medulloblastomas in 80% of children yet also causes cell death in healthy neurons, resulting in a permanent and devastating loss of IQ. Likewise, children commonly experience dose-limiting cardiotoxicity from doxorubicin treatment. These treatments are comparatively well tolerated in adults yet the basis for this dramatic contrast in sensitivity is unknown. Both radiation and cytotoxic chemotherapies can induce an apoptotic cell death, prompting us to hypothesize that apoptosis may be regulated in a fundamentally different manner in children versus adults. By testing the functional state of the apoptotic pathway in tissues with BH3 Profiling, we found that healthy brain, heart and kidney tissues from young mice are extremely sensitive to pro-apoptotic signals and are therefore “primed for apoptosis.” In stark contrast, these same tissues are completely insensitive to even saturating amounts of pro-apoptotic signals in adult mice. Apoptosis could be triggered in adult brain, heart or kidney cells only when complemented with exogenous BAX protein suggesting that these tissues lack sufficient levels of BAX or BAK for mitochondrial permeabilization and are thus “incompetent for apoptosis.” Immunoblotting revealed that BAX and BAK protein levels are strongly downregulated in these tissues during postnatal development and become nearly undetectable by adulthood, along with several other components of the apoptotic machinery. Parallel studies with spleen and bone marrow demonstrated high levels of apoptotic priming in young animals which continued into adulthood, highlighting the organ-specific nature of apoptosis regulation. In agreement with these findings, we observed apoptotic cell death in response to radiation damage in the brain, heart and kidneys of early postnatal but not adult mice. Likewise, the extent of doxorubicin-induced damage to cardiac tissue correlated strongly with changes in apoptotic priming and competence. Furthermore, we utilized BAX and/or BAK knockout mice to characterize the pivotal roles of these proteins in regulating apoptotic competence and damage responses in somatic tissues. We extended these findings to humans by BH3 profiling normal brain tissue and confirmed that brain tissue in young children is primed to undergo apoptosis while in adults it is apoptotically incompetent. Finally, we identified the epigenetic mechanisms that modulate apoptotic pathways during postnatal development. Our findings elucidate the molecular mechanisms responsible for the devastating vital organ sensitivity to damage in children and a strategy for its prevention. Citation Format: Kristopher A. Sarosiek, Michael Ziller, Cameron Fraser, Patrick Bhola, Jeremy Ryan, Jing Deng, Brian Jian, Marti Goldenberg, Joseph Madsen, Ruben Carrasco, Shenandoah Robinson, Javid Moslehi, Anthony Letai. Apoptotic priming is regulated by a developmental program and predisposes children to therapy-induced toxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4728. doi:10.1158/1538-7445.AM2015-4728

51INTARGETING THE BCL-2 PATHWAY

ABSTRACT Many, if not most, anti-cancer agents kill cancer cells via the mitochondrial pathway of apoptosis. We have investigated ways that we can predict whether a particular agent will active apoptotic signaling on a personalized basis. We have found that we can predict response to conventional chemotherapy and to BCL-2 inhibition based on BH3 Profiling. BH3 Profiling is a functional assay that measures, on a single cell basis, mitochondrial sensitivity to BH3 peptides. As such, it can measure how primed a cell is to apoptosis. In a more recent application, we have combined BH3 Profiling with brief ex vivo exposures of patient cancer cells to drugs. We have found that this provides very specific information predicting whether an individuals cancer cells will respond to an individual agent. It can be mutliplexed so that many drugs can be simultaneously evaluated, even in combination. This approach, which we call Dynamic BH3 Profiling, can be applied as a personalization strategy for cancer patients. Most predictive biomarker approaches focus on the study of components of dead, non-functional cells. BH3 Profiling demonstrates the great amount of useful information that can be obtained by the study of strategic perturbations of functional cells. Disclosure: A. Letai: Dr. Letai has received fees for adviding AbbVie. Dr. Letais laborotary has received funding to support research from AbbVie. All other authors have declared no conflicts of interest.