Functional genomic screens with death rate analyses reveal mechanisms of drug action.

A common approach for understanding how drugs induce their therapeutic effects is to identify the genetic determinants of drug sensitivity. Because 'chemo-genetic profiles' are performed in a pooled format, inference of gene function is subject to several confounding influences related to variation in growth rates between clones. In this study, we developed Method for Evaluating Death Using a Simulation-assisted Approach (MEDUSA), which uses time-resolved measurements, along with model-driven constraints, to reveal the combination of growth and death rates that generated the observed drug response. MEDUSA is uniquely effective at identifying death regulatory genes. We apply MEDUSA to characterize DNA damage-induced lethality in the presence and absence of p53. Loss of p53 switches the mechanism of DNA damage-induced death from apoptosis to a non-apoptotic death that requires high respiration. These findings demonstrate the utility of MEDUSA both for determining the genetic dependencies of lethality and for revealing opportunities to potentiate chemo-efficacy in a cancer-specific manner.

Targeting Galectin 3 illuminates its contributions to the pathology of uterine serous carcinoma.

BACKGROUND: Uterine serous cancer (USC) comprises around 10% of all uterine cancers. However, USC accounts for approximately 40% of uterine cancer deaths, which is attributed to tumor aggressiveness and limited effective treatment. Galectin 3 (Gal3) has been implicated in promoting aggressive features in some malignancies. However, Gal3's role in promoting USC pathology is lacking. METHODS: We explored the relationship between LGALS3 levels and prognosis in USC patients using TCGA database, and examined the association between Gal3 levels in primary USC tumors and clinical-pathological features. CRISPR/Cas9-mediated Gal3-knockout (KO) and GB1107, inhibitor of Gal3, were employed to evaluate Gal3's impact on cell function. RESULTS: TCGA analysis revealed a worse prognosis for USC patients with high LGALS3. Patients with no-to-low Gal3 expression in primary tumors exhibited reduced clinical-pathological tumor progression. Gal3-KO and GB1107 reduced cell proliferation, stemness, adhesion, migration, and or invasion properties of USC lines. Furthermore, Gal3-positive conditioned media (CM) stimulated vascular tubal formation and branching and transition of fibroblast to cancer-associated fibroblast compared to Gal3-negative CM. Xenograft models emphasized the significance of Gal3 loss with fewer and smaller tumors compared to controls. Moreover, GB1107 impeded the growth of USC patient-derived organoids. CONCLUSION: These findings suggest inhibiting Gal3 may benefit USC patients.

In situ architecture of Opa1-dependent mitochondrial cristae remodeling.

Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria, while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and show a compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

Role of intrinsic apoptosis in environmental exposure health outcomes.

Environmental exposures are linked to diseases of high public health concern, including cancer, neurodegenerative disorders, and autoimmunity. These diseases are caused by excessive or insufficient cell death, prompting investigation of mechanistic links between environmental toxicants and dysregulation of cell death pathways, including apoptosis. This review describes how legacy and emerging environmental exposures target the intrinsic apoptosis pathway to potentially drive pathogenesis. Recent discoveries reveal that dynamic regulation of apoptosis may heighten the vulnerability of healthy tissues to exposures in children, and that apoptotic signaling can guide immune responses, tissue repair, and tumorigenesis. Understanding how environmental toxicants dysregulate apoptosis will uncover opportunities to deploy apoptosis-modulating agents for the treatment or prevention of exposure-linked diseases.

Pulsed ultrasound promotes secretion of anti-inflammatory extracellular vesicles from skeletal myotubes via elevation of intracellular calcium level.

The regulation of inflammatory responses is an important intervention in biological function and macrophages play an essential role during inflammation. Skeletal muscle is the largest organ in the human body and releases various factors which mediate anti-inflammatory/immune modulatory effects. Recently, the roles of extracellular vesicles (EVs) from a large variety of cells are reported. In particular, EVs released from skeletal muscle are attracting attention due to their therapeutic effects on dysfunctional organs and tissues. Also, ultrasound (US) promotes release of EVs from skeletal muscle. In this study, we investigated the output parameters and mechanisms of US-induced EV release enhancement and the potential of US-treated skeletal muscle-derived EVs in the regulation of inflammatory responses in macrophages. High-intensity US (3.0 W/cm2) irradiation increased EV secretion from C2C12 murine muscle cells via elevating intracellular Ca2+ level without negative effects. Moreover, US-induced EVs suppressed expression levels of pro-inflammatory factors in macrophages. miRNA sequencing analysis revealed that miR-206-3p and miR-378a-3p were especially abundant in skeletal myotube-derived EVs. In this study we demonstrated that high-intensity US promotes the release of anti-inflammatory EVs from skeletal myotubes and exert anti-inflammatory effects on macrophages.

Tuning ultrasmall theranostic nanoparticles for MRI contrast and radiation dose amplification.

Background: The introduction of magnetic resonance (MR)-guided radiation treatment planning has opened a new space for theranostic nanoparticles to reduce acute toxicity while improving local control. In this work, second-generation AGuIX® nanoparticles (AGuIX-Bi) are synthesized and validated. AGuIX-Bi are shown to maintain MR positive contrast while further amplifying the radiation dose by the replacement of some Gd3+ cations with higher Z Bi3+. These next-generation nanoparticles are based on the AGuIX® platform, which is currently being evaluated in multiple Phase II clinical trials in combination with radiotherapy. Methods: In this clinically scalable methodology, AGuIX® is used as an initial chelation platform to exchange Gd3+ for Bi3+. AGuIX-Bi nanoparticles are synthesized with three ratios of Gd/Bi, each maintaining MR contrast while further amplifying radiation dose relative to Bi3+. Safety, efficacy, and theranostic potential of the nanoparticles were evaluated in vitro and in vivo in a human non-small cell lung cancer model. Results: We demonstrated that increasing Bi3+ in the nanoparticles is associated with more DNA damage and improves in vivo efficacy with a statistically significant delay in tumor growth and 33% complete regression for the largest Bi/Gd ratio tested. The addition of Bi3+ by our synthetic method leads to nanoparticles that present slightly altered pharmacokinetics and lengthening of the period of high tumor accumulation with no observed evidence of toxicity. Conclusions: We confirmed the safety and enhanced efficacy of AGuIX-Bi with radiation therapy at the selected ratio of 30Gd/70Bi. These results provide crucial evidence towards patient translation.

Radiotherapy-Induced Neurocognitive Impairment Is Driven by Heightened Apoptotic Priming in Early Life and Prevented by Blocking BAX.

Although external beam radiotherapy (xRT) is commonly used to treat central nervous system (CNS) tumors in patients of all ages, young children treated with xRT frequently experience life-altering and dose-limiting neurocognitive impairment (NI) while adults do not. The lack of understanding of mechanisms responsible for these differences has impeded the development of neuroprotective treatments. Using a newly developed mouse model of xRT-induced NI, we found that neurocognitive function is impaired by ionizing radiation in a dose- and age-dependent manner, with the youngest animals being most affected. Histologic analysis revealed xRT-driven neuronal degeneration and cell death in neurogenic brain regions in young animals but not adults. BH3 profiling showed that neural stem and progenitor cells, neurons, and astrocytes in young mice are highly primed for apoptosis, rendering them hypersensitive to genotoxic damage. Analysis of single-cell RNA sequencing data revealed that neural cell vulnerability stems from heightened expression of proapoptotic genes including BAX, which is associated with developmental and mitogenic signaling by MYC. xRT induced apoptosis in primed neural cells by triggering a p53- and PUMA-initiated, proapoptotic feedback loop requiring cleavage of BID and culminating in BAX oligomerization and caspase activation. Notably, loss of BAX protected against apoptosis induced by proapoptotic signaling in vitro and prevented xRT-induced apoptosis in neural cells in vivo as well as neurocognitive sequelae. On the basis of these findings, preventing xRT-induced apoptosis specifically in immature neural cells by blocking BAX, BIM, or BID via direct or upstream mechanisms is expected to ameliorate NI in pediatric patients with CNS tumor. SIGNIFICANCE: Age- and differentiation-dependent apoptotic priming plays a pivotal role in driving radiotherapy-induced neurocognitive impairment and can be targeted for neuroprotection in pediatric patients.

Endogenous and imposed determinants of apoptotic vulnerabilities in cancer.

The intrinsic apoptosis pathway is controlled by the BCL-2 family of proteins. Although the pro-survival members of this family can help cancer cells evade apoptosis, they may also produce apoptotic vulnerabilities that can potentially be exploited therapeutically. Apoptotic vulnerabilities can be driven by endogenous factors including altered genetics, signaling, metabolism, structure and lineage or differentiation state as well as imposed factors, the most prominent being exposure to anti-cancer agents. The recent development of BH3 mimetics that inhibit pro-survival BCL-2 family proteins has allowed these apoptotic vulnerabilities to be targeted with demonstrable clinical success. Here, we review the key concepts that are vital for understanding, uncovering, and exploiting apoptotic vulnerabilities in cancer for the potential improvement of patient outcomes.

Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.

In situ architecture of Opa1-dependent mitochondrial cristae remodeling.

Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

p53 controls choice between apoptotic and non-apoptotic death following DNA damage.

DNA damage can activate apoptotic and non-apoptotic forms of cell death; however, it remains unclear what features dictate which type of cell death is activated. We report that p53 controls the choice between apoptotic and non-apoptotic death following exposure to DNA damage. In contrast to the conventional model, which suggests that p53-deficient cells should be resistant to DNA damage-induced cell death, we find that p53-deficient cells die at high rates following DNA damage, but exclusively using non-apoptotic mechanisms. Our experimental data and computational modeling reveal that non-apoptotic death in p53-deficient cells has not been observed due to use of assays that are either insensitive to cell death, or that specifically score apoptotic cells. Using functional genetic screening - with an analysis that enables computational inference of the drug-induced death rate - we find in p53-deficient cells that DNA damage activates a mitochondrial respiration-dependent form of cell death, called MPT-driven necrosis. Cells deficient for p53 have high basal respiration, which primes MPT-driven necrosis. Finally, using metabolite profiling, we identified mitochondrial activity-dependent metabolic vulnerabilities that can be targeted to potentiate the lethality of DNA damage specifically in p53-deficient cells. Our findings reveal how the dual functions of p53 in regulating mitochondrial activity and the DNA damage response combine to facilitate the choice between apoptotic and non-apoptotic death.

Peptides from human BNIP5 and PXT1 and non-native binders of pro-apoptotic BAK can directly activate or inhibit BAK-mediated membrane permeabilization.

Apoptosis is important for development and tissue homeostasis, and its dysregulation can lead to diseases, including cancer. As an apoptotic effector, BAK undergoes conformational changes that promote mitochondrial outer membrane disruption, leading to cell death. This is termed "activation" and can be induced by peptides from the human proteins BID, BIM, and PUMA. To identify additional peptides that can regulate BAK, we used computational protein design, yeast surface display screening, and structure-based energy scoring to identify 10 diverse new binders. We discovered peptides from the human proteins BNIP5 and PXT1 and three non-native peptides that activate BAK in liposome assays and induce cytochrome c release from mitochondria. Crystal structures and binding studies reveal a high degree of similarity among peptide activators and inhibitors, ruling out a simple function-determining property. Our results shed light on the vast peptide sequence space that can regulate BAK function and will guide the design of BAK-modulating tools and therapeutics.

Heightened apoptotic priming of vascular cells across tissues and life span predisposes them to cancer therapy-induced toxicities.

Although major organ toxicities frequently arise in patients treated with cytotoxic or targeted cancer therapies, the mechanisms that drive them are poorly understood. Here, we report that vascular endothelial cells (ECs) are more highly primed for apoptosis than parenchymal cells across many adult tissues. Consequently, ECs readily undergo apoptosis in response to many commonly used anticancer agents including cytotoxic and targeted drugs and are more sensitive to ionizing radiation and BH3 mimetics than parenchymal cells in vivo. Further, using differentiated isogenic human induced pluripotent stem cell models of ECs and vascular smooth muscle cells (VSMCs), we find that these vascular cells exhibit distinct drug toxicity patterns, which are linked to divergent therapy-induced vascular toxicities in patients. Collectively, our results demonstrate that vascular cells are highly sensitive to apoptosis-inducing stress across life span and may represent a "weakest link" vulnerability in multiple tissues for development of toxicities.

Reciprocal effects of mTOR inhibitors on pro-survival proteins dictate therapeutic responses in tuberous sclerosis complex.

mTORC1 is aberrantly activated in cancer and in the genetic tumor syndrome tuberous sclerosis complex (TSC), which is caused by loss-of-function mutations in the TSC complex, a negative regulator of mTORC1. Clinically approved mTORC1 inhibitors, such as rapamycin, elicit a cytostatic effect that fails to eliminate tumors and is rapidly reversible. We sought to determine the effects of mTORC1 on the core regulators of intrinsic apoptosis. In TSC2-deficient cells and tumors, we find that mTORC1 inhibitors shift cellular dependence from MCL-1 to BCL-2 and BCL-XL for survival, thereby altering susceptibility to BH3 mimetics that target specific pro-survival BCL-2 proteins. The BCL-2/BCL-XL inhibitor ABT-263 synergizes with rapamycin to induce apoptosis in TSC-deficient cells and in a mouse tumor model of TSC, resulting in a more complete and durable response. These data expose a therapeutic vulnerability in regulation of the apoptotic machinery downstream of mTORC1 that promotes a cytotoxic response to rapamycin.

Exploiting endogenous and therapy-induced apoptotic vulnerabilities in immunoglobulin light chain amyloidosis with BH3 mimetics.

Immunoglobulin light chain (AL) amyloidosis is an incurable hematologic disorder typically characterized by the production of amyloidogenic light chains by clonal plasma cells. These light chains misfold and aggregate in healthy tissues as amyloid fibrils, leading to life-threatening multi-organ dysfunction. Here we show that the clonal plasma cells in AL amyloidosis are highly primed to undergo apoptosis and dependent on pro-survival proteins MCL-1 and BCL-2. Notably, this MCL-1 dependency is indirectly targeted by the proteasome inhibitor bortezomib, currently the standard of care for this disease and the related plasma cell disorder multiple myeloma, due to upregulation of pro-apoptotic Noxa and its inhibitory binding to MCL-1. BCL-2 inhibitors sensitize clonal plasma cells to multiple front-line therapies including bortezomib, dexamethasone and lenalidomide. Strikingly, in mice bearing AL amyloidosis cell line xenografts, single agent treatment with the BCL-2 inhibitor ABT-199 (venetoclax) produces deeper remissions than bortezomib and triples median survival. Mass spectrometry-based proteomic analysis reveals rewiring of signaling pathways regulating apoptosis, proliferation and mitochondrial metabolism between isogenic AL amyloidosis and multiple myeloma cells that divergently alter their sensitivity to therapies. These findings provide a roadmap for the use of BH3 mimetics to exploit endogenous and induced apoptotic vulnerabilities in AL amyloidosis.

Small molecule SJ572946 activates BAK to initiate apoptosis.

Poration of the outer mitochondrial membrane by the effector BCL-2 proteins BAK and BAX initiates apoptosis. BH3-only initiators BID and BIM trigger conformational changes in BAK and BAX transforming them from globular dormant proteins to oligomers of the apoptotic pores. Small molecules that can directly activate effectors are being sought for applications in cancer treatment. Here, we describe the small molecule SJ572946, discovered in a fragment-based screen that binds to the activation groove of BAK and selectively triggers BAK activation over that of BAX in liposome and mitochondrial permeabilization assays. SJ572946 independently kills BAK-expressing BCL2allKO HCT116 cells revealing on target cellular activity. In combination with apoptotic inducers and BH3 mimetics, SJ572946 kills experimental cancer cell lines. SJ572946 also cooperates with the endogenous BAK activator BID in activating a misfolded BAK mutant substantially impaired in activation. SJ572946 is a proof-of-concept tool for probing BAK-mediated apoptosis in preclinical cancer research.

A Mesenchymal Tumor Cell State Confers Increased Dependency on the BCL-XL Antiapoptotic Protein in Kidney Cancer.

PURPOSE: Advanced/metastatic forms of clear-cell renal cell carcinomas (ccRCC) have limited therapeutic options. Genome-wide genetic screens have identified cellular dependencies in many cancers. Using the Broad Institute/Novartis combined short hairpin RNA (shRNA) dataset, and cross-validation with the CRISPR/Cas9 DepMap (21Q3) dataset, we sought therapeutically actionable dependencies in kidney lineage cancers. EXPERIMENTAL DESIGN: We identified preferential genetic dependencies in kidney cancer cells versus other lineages. BCL2L1, which encodes the BCL-XL antiapoptotic protein, scored as the top actionable dependency. We validated this finding using genetic and pharmacologic tools in a panel of ccRCC cell lines. Select BCL-XL-dependent (versus independent) cell lines were then transcriptionally profiled to identify biomarkers and mechanistic drivers of BCL-XL dependence. Cell-based studies (in vitro and in vivo) and clinical validations were used to address physiologic relevance. RESULTS: Inactivation of BCL-XL, but not BCL-2, led to fitness defects in renal cancer cells, and sensitized them to chemotherapeutics. Transcriptomic profiling identified a "BCL-XL dependency" signature, including an elevated mesenchymal gene signature. A mesenchymal state was both necessary and sufficient to confer increased BCL-XL dependence. The "BCL-XL dependency" signature was observed in approximately 30% of human ccRCCs, which were also associated with worse clinical outcomes. Finally, an orally bioavailable BCL-XL inhibitor, A-1331852, showed antitumor efficacy in vivo. CONCLUSIONS: Our studies uncovered an unexpected link between cell state and BCL-XL dependence in ccRCC. Therapeutic agents that specifically target BCL-XL are available. Our work justifies testing the utility of BCL-XL blockade to target, likely, a clinically aggressive subset of human kidney cancers. See related commentary by Wang et al., p. 4600.

Age-dependent regulation of SARS-CoV-2 cell entry genes and cell death programs correlates with COVID-19 severity.

Novel coronavirus disease 2019 (COVID-19) severity is highly variable, with pediatric patients typically experiencing less severe infection than adults and especially the elderly. The basis for this difference is unclear. We find that mRNA and protein expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, increases with advancing age in distal lung epithelial cells. However, in humans, ACE2 expression exhibits high levels of intra- and interindividual heterogeneity. Further, cells infected with SARS-CoV-2 experience endoplasmic reticulum stress, triggering an unfolded protein response and caspase-mediated apoptosis, a natural host defense system that halts virion production. Apoptosis of infected cells can be selectively induced by treatment with apoptosis-modulating BH3 mimetic drugs. Notably, epithelial cells within young lungs and airways are more primed to undergo apoptosis than those in adults, which may naturally hinder virion production and support milder COVID-19 severity.

Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics.

Cancer cells have differential metabolic dependencies compared to their nonmalignant counterparts. However, few metabolism-targeting compounds have been successful in clinical trials. Here, we investigated the metabolic vulnerabilities of triple-negative breast cancer (TNBC), particularly those metabolic perturbations that increased mitochondrial apoptotic priming and sensitivity to BH3 mimetics (drugs that antagonize antiapoptotic proteins). We used high-throughput dynamic BH3 profiling (HT-DBP) to screen a library of metabolism-perturbing small molecules, which revealed inhibitors of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) as top candidates. In some TNBC cells but not in nonmalignant cells, NAMPT inhibitors increased overall apoptotic priming and induced dependencies on specific antiapoptotic BCL-2 family members. Treatment of TNBC cells with NAMPT inhibitors sensitized them to subsequent treatment with BH3 mimetics. The combination of a NAMPT inhibitor (FK866) and an MCL-1 antagonist (S63845) reduced tumor growth in a TNBC patient-derived xenograft model in vivo. We found that NAMPT inhibition reduced NAD+ concentrations below a critical threshold that resulted in depletion of adenine, which was the metabolic trigger that primed TNBC cells for apoptosis. These findings demonstrate a close interaction between metabolic and mitochondrial apoptotic signaling pathways and reveal that exploitation of a tumor-specific metabolic vulnerability can sensitize some TNBC to BH3 mimetics.