2.1.1 Intrinsic (mitochondrial) pathway

The intrinsic pathway of apoptosis is primarily regulated by mitochondrial signals and is critically dependent on the balance between proapoptotic and antiapoptotic members of the Bcl-2 family of proteins (Figure 1A). This pathway is typically triggered by various forms of cellular stress, including DNA damage, oxidative stress, and growth factor deprivation.^{24, 25}

2.1.2 Extrinsic (death receptor) pathway

The initiation of the extrinsic pathway occurs when extracellular death ligands bind to their respective death receptors on the cell surface (Figure 1B), a process essential for the immune system in eliminating infected or transformed cells.^36-38

2.2.1 Characteristics of necrosis

Necrosis is typically seen as a type of cell death that occurs when there is sudden damage to the cells. ²⁴ This kind of cell death is identified by a breakdown in membrane integrity, uncontrolled release of cell contents, and inflammation that follows.⁴⁶ Unlike apoptosis, necrosis does not involve specific signaling pathways, and it typically results from factors such as trauma, infection, or ischemia. Morphologically, necrotic cells exhibit swelling (oncosis), organelle disruption, and eventual rupture of the plasma membrane. The release of intracellular components into the extracellular space due to these events triggers an inflammatory response from the immune system, which can lead to tissue damage and disease progression because of the uncontrolled nature of necrosis and the associated inflammation.⁴⁸

2.2.2 Regulated necrosis: necroptosis

Necroptosis is a form of programmed necrosis that shares morphological features with necrosis but is executed through regulated signaling pathways. This process can be triggered under conditions where apoptosis is inhibited, serving as a backup mechanism to ensure cell death.^49-51 Necroptosis plays a crucial role in various physiological and pathological contexts, including development, immune response, and disease.^52-54 The molecular machinery governing necroptosis involves a well-coordinated interaction between RIPKs and MLKL.⁵⁵ The key steps in the necroptotic pathway are as follows: ① RIPK1 activation: Necroptosis is often initiated by the activation of death receptors, such as TNF receptor 1 (TNFR1), which can recruit RIPK1 through its DD. Upon activation, RIPK1 undergoes autophosphorylation and forms a complex with RIPK3, known as the necrosome.^{56, 57} ② RIPK3 activation: Within the necrosome, RIPK3 is phosphorylated by RIPK1. This phosphorylation event is critical for the downstream signaling cascade that leads to necroptosis. RIPK3, in turn, recruits and phosphorylates MLKL.^{58, 59} ③ MLKL activation and membrane disruption: Phosphorylated MLKL undergoes a conformational change, allowing it to oligomerize and translocate to the plasma membrane. Once at the membrane, MLKL disrupts membrane integrity by forming pores, leading to cell swelling, membrane rupture, and ultimately necrotic cell death (Figure 2A).⁶⁰

To sum up, necrosis and necroptosis are two different but related types of cell death. Whereas necrosis occurs from sudden damage to cells, necroptosis is a controlled form of cell death involving RIPK1, RIPK3, and MLKL.^{61, 62} Understanding the molecular mechanisms behind these processes can help us better comprehend their impact on health and disease, and potentially identify new treatment targets for disorders marked by abnormal cell death.

2.3.1 Mechanisms of autophagy

Autophagy, a catabolic process involving the breakdown of cellular components through lysosomal machinery, is derived from the Greek words “self” and “eating.”⁶⁵ The process can be divided into several key stages: ① Initiation: Autophagy starts with the activation of the ULK1 complex, which consists of ULK1, FIP200, ATG13, and ATG101, and is regulated by nutrient and energy sensors like mTOR and AMP-activated protein kinase (AMPK).⁶⁶ ② Nucleation: The activated ULK1 complex triggers the formation of the phagophore, a double-membraned structure, with the help of the class III PI3K complex containing VPS34, Beclin-1, and ATG14L, creating phosphatidylinositol 3-phosphate (PI3P) to recruit other autophagy-related proteins.⁶⁶ ③ Elongation and maturation: The phagophore expands to engulf cytoplasmic material, with the assistance of the ATG12–ATG5–ATG16L complex and the LC3–PE conjugation system. LC3-II is crucial for membrane expansion and cargo recognition.⁶⁷ ④ The autophagosome merges with lysosomes to form an autolysosome, where lysosomal hydrolases break down the sequestered material into basic biomolecules that can be recycled for reuse in the cytoplasm (Figure 2B).⁶⁸

2.3.2 Role of autophagy in cancer

Autophagy plays a complex and context-dependent role in cancer, acting as a double-edged sword with both tumor-suppressive and tumor-promoting effects.⁶⁹ In the early stages of cancer development, autophagy can act as a tumor suppressor.⁷⁰ By degrading damaged organelles and proteins, autophagy prevents the accumulation of cellular damage and maintains genomic stability. This process reduces oxidative stress and inhibits chronic inflammation, both of which are associated with oncogenesis. Furthermore, autophagy can induce autophagic cell death in cancer cells, especially under conditions of metabolic stress or treatment with chemotherapeutic agents.^{69, 71} Paradoxically, in established tumors, autophagy often supports cancer cell survival and growth. Tumor cells exploit autophagy to withstand metabolic stress, hypoxia, and nutrient deprivation commonly found in the TME. By recycling intracellular components, autophagy provides essential substrates for energy production and biosynthesis, promoting tumor cell proliferation and survival. Additionally, autophagy can contribute to therapy resistance by enabling cancer cells to survive cytotoxic treatments.^{72, 73} Given its dual role, targeting autophagy in cancer therapy requires a nuanced approach. Inhibiting autophagy may be beneficial in established tumors where autophagy supports survival and growth. Several clinical trials are investigating autophagy inhibitors, such as hydroxychloroquine (HCQ), in combination with conventional therapies.⁷⁴ Conversely, enhancing autophagy could be a strategy to induce autophagic cell death in certain cancer types or sensitize tumors to chemotherapy and radiation.⁷⁵

In conclusion, autophagy is a multifaceted process that plays critical roles in cellular homeostasis and disease. Its involvement in cancer is particularly complex, encompassing both tumor-suppressive and tumor-promoting functions.⁷⁶ Understanding the context-dependent nature of autophagy in cancer is essential for developing effective therapeutic strategies that harness or inhibit this process to benefit patients.

2.4.1 Iron-dependent lipid peroxidation

The accumulation of lipid peroxides drives ferroptosis, which can become toxic when levels are excessive, and is reliant on iron, a transition metal that triggers the production of reactive oxygen species (ROS) via the Fenton reaction.^{81, 82} The key steps in iron-dependent lipid peroxidation include iron uptake and storage, ROS generation, and lipid peroxidation.⁸³ Iron is taken up by cells via transferrin receptor-mediated endocytosis of transferrin-bound iron, which is then released from endosomes and stored in the cytosol in a labile iron pool or sequestered in ferritin. Free iron in the labile iron pool can participate in the Fenton reaction, converting hydrogen peroxide (H₂O₂) into highly reactive hydroxyl radicals (•OH), which initiate lipid peroxidation by abstracting hydrogen atoms from polyunsaturated fatty acids in membrane phospholipids. The initial lipid radicals formed are further oxidized to lipid peroxides, which, if not adequately detoxified, can decompose into reactive aldehydes and propagate further lipid peroxidation, leading to the loss of membrane integrity and cell death.^84-86

2.4.2 Regulation by GPX4 and system Xc⁻

Critical antioxidant systems are involved in regulating ferroptosis to prevent the buildup of lipid peroxides, with two main regulators being glutathione peroxidase 4 (GPX4) and the cystine/glutamate antiporter system Xc⁻.⁸⁷ GPX4, a selenoprotein, is essential in protecting cells from ferroptosis by converting lipid hydroperoxides (LOOH) into alcohols, thus stopping lipid peroxidation.^{88, 89} GPX4 requires glutathione (GSH) as a substrate to exert its peroxidase activity. It specifically reduces LOOH within membranes to nontoxic lipid alcohols, thus inhibiting the chain reaction of lipid peroxidation. The activity of GPX4 is directly linked to the intracellular levels of GSH, which in turn is synthesized from cysteine. Therefore, the availability of cysteine is crucial for maintaining GPX4 activity and preventing ferroptosis.⁹⁰ System Xc⁻ is a plasma membrane transporter composed of two subunits, SLC7A11 and SLC3A2, which mediates the exchange of extracellular cystine and intracellular glutamate. By importing cystine into the cell in exchange for glutamate, system Xc⁻ facilitates the synthesis of GSH. Cystine is reduced to cysteine inside the cell, which is then used to synthesize GSH. The activity of system Xc⁻ is influenced by various factors, including oxidative stress and nutrient availability. ^{91, 92}

In summary, ferroptosis is a controlled form of cell death triggered by iron-induced lipid peroxidation. The susceptibility of cells to ferroptosis depends on the balance between the production of lipid peroxides and their removal by antioxidant mechanisms like GPX4 and system Xc⁻ (Figure 2C). Understanding the molecular mechanisms underlying ferroptosis provides valuable insights into its role in various diseases and offers potential therapeutic targets for conditions where ferroptosis plays a critical role.

2.5.1 Inflammatory caspases

Inflammatory caspases, including caspase-1, -4, -5, and -11, are crucial for initiating and carrying out pyroptosis.^{96, 97} Caspase-1 is activated by pathogen-associated molecular patterns and DAMPs through inflammasomes like the NLRP3 inflammasome.^{98, 99} Once activated, it converts pro-IL-1β and pro-IL-18 into IL-1β and IL-18, and triggers pyroptosis by cleaving GSDMD.¹⁰⁰ On the other hand, caspase-4, -5, and -11 are activated by intracellular lipopolysaccharides without an inflammasome, also leading to pyroptosis by cleaving GSDMD. While they do not directly process pro-IL-1β and pro-IL-18, their activation can enhance the inflammatory response through secondary pathways.^{101, 102}

2.5.2 Formation of gasdermin pores

The main mechanism behind pyroptosis involves GSDMD, a protein from the gasdermin family, creating pores in the cell membrane.¹⁰³ When activated, inflammatory caspases break down GSDMD at a specific location, separating its pore-forming domain from its inhibitory domain. This separation releases the pore-forming domain, which moves to the cell membrane and binds to phospholipids. Multiple pore-forming domains then come together to create large pores, disrupting the cell membrane and causing cell swelling, membrane rupture, and the release of cell contents. These pores allow proinflammatory cytokines like IL-1β and IL-18 to be released, promoting inflammation by attracting and activating immune cells. Ultimately, the cell membrane ruptures, releasing DAMPs and amplifying the immune response to further contribute to inflammation.^{96, 104, 105}

In summary, pyroptosis is a type of cell death that involves inflammation, triggered by activating inflammatory caspases and creating gasdermin pores. Inflammatory caspases like caspase-1, -4, -5, and -11 are essential for detecting harmful signals, causing the breakdown of GSDMD. The GSDMD N-terminal pieces generate pores in the cell membrane, leading to cell rupture and the discharge of proinflammatory substances (Figure 2D). Understanding the mechanisms of pyroptosis provides insights into its roles in various diseases, particularly those involving infection and inflammation, and offers potential targets for therapeutic intervention.

3.1.1 Mutations in key regulators

3.1.2 Epigenetic modifications

Histone modification

Histone proteins, wrapped around DNA, can be modified after translation in various ways, such as methylation, acetylation, phosphorylation, and ubiquitination, which can affect chromatin structure and gene expression. In relation to cell death, histone acetylation usually promotes the activation of proapoptotic genes, while deacetylation by histone deacetylases can suppress these genes, preventing apoptosis.¹¹⁶ Similarly, histone methylation can either enhance or inhibit gene expression, depending on the specific residues impacted; for example, trimethylation of histone H3 lysine 4 (H3K4me3) is connected to gene activation, while trimethylation of histone H3 lysine 27 (H3K27me3) is associated with gene repression. Alterations in these histone modifications can have a significant impact on the regulation of genes involved in cell death processes.^{116, 117}

In summary, both genetic and epigenetic factors play crucial roles in regulating cell death. Mutations in key regulators such as TP53 and Bcl-2 family genes can disrupt normal cell death mechanisms, contributing to diseases like cancer. Epigenetic modifications, including DNA methylation and histone modifications, further modulate the expression of genes involved in cell death, influencing cellular responses to stress and damage (Figure 3A). Having a grasp on these genetic and epigenetic factors gives us valuable information about how cell death works and presents possible targets for treating different diseases.

3.2.1 PI3K/AKT/mTOR pathway

The PI3K/AKT/mTOR pathway plays a crucial role in regulating cell survival, growth, proliferation, and metabolism, with its disruption being common in cancer and other diseases.¹¹⁸ Growth factors, cytokines, and other external signals can activate PI3K through receptor tyrosine kinases (RTKs) or G protein-coupled receptors. Upon activation, PI3K phosphorylates phosphatidylinositol (4,5)-bisphosphate (PIP2) to produce phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a lipid second messenger. PIP3 then recruits AKT to the cell membrane, where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 and mTOR complex 2 (mTORC2). Once activated, AKT supports cell survival and growth by phosphorylating and inhibiting proapoptotic substances like BAD and FOXO, while also enhancing the activity of prosurvival proteins such as Murine double minute 2. mTOR, a serine/threonine kinase, operates in two different complexes known as mTORC1 and mTORC2. AKT directly activates mTORC1 by inhibiting the TSC1/TSC2 complex, which negatively regulates mTORC1. mTORC1 promotes protein synthesis, cell growth, and survival by phosphorylating downstream targets like S6K1 and 4E-BP1, while mTORC2 phosphorylates AKT, further enhancing its activity.^119-121

3.2.2 MAPK/ERK pathway

The MAPK/ERK pathway plays a crucial role in controlling cell proliferation, differentiation, and survival, with key components including rat sarcoma (RAS), rapidly accelerated fibrosarcoma (RAF), mitogen-activated protein kinase kinase (MEK), and ERK. Activation occurs when external signals stimulate RTKs, leading to RAS activation and subsequent recruitment of RAF kinases. RAF then activates MEK, which in turn activates ERK. ERK travels to the nucleus and activates transcription factors like ELK1 and c-FOS, resulting in gene expression related to cell growth and survival. ERK also contributes to cell survival by inhibiting proapoptotic proteins like BAD and increasing the levels of antiapoptotic proteins such as Bcl-2 and MCL-1. Nevertheless, prolonged activation of the MAPK/ERK pathway under stress conditions can trigger cell death by activating proapoptotic genes.^{122, 123}

3.2.3 NF-κB signaling

The NF-κB pathway plays a crucial role in regulating immune responses, inflammation, and cell survival.¹²⁴ The NF-κB family consists of transcription factors like p65 (RelA), p50, and c-Rel, which combine to form different homo- and heterodimers. Activation of NF-κB can occur in response to various stimuli, such as proinflammatory cytokines like TNF-α and IL-1, microbial products, and stress signals. These stimuli trigger the IκB kinase (IKK) complex to phosphorylate and break down IκB proteins that inhibit NF-κB. The breakdown of IκB enables NF-κB dimers to move into the nucleus. In the nucleus, NF-κB binds to specific DNA sequences and activates the transcription of genes associated with inflammation, immune response, cell survival, and cell proliferation.^{125, 126} This pathway promotes cell survival by elevating the expression of antiapoptotic genes like Bcl-2, Bcl-xL, and x-linked inhibitor of apoptosis protein, as well as genes involved in cell proliferation such as c-MYC. Furthermore, NF-κB activation can lead to the production of proinflammatory cytokines and chemokines, which can either support cell survival or contribute to cell death during chronic inflammation.^{124, 127, 128}

In essence, the PI3K/AKT/mTOR, MAPK/ERK, and NF-κB signaling pathways play a vital role in controlling cell fate determinations by incorporating external signals to regulate the functions of different proteins connected to cell survival and apoptosis. Disorders in these pathways are frequently linked to illnesses like cancer, underscoring their significance as possible treatment targets. Comprehending the complexities of these signaling systems offers valuable knowledge about the processes that oversee cell survival and death.

3.3.1 Hypoxia and nutrient deprivation

Hypoxia (low oxygen levels) and nutrient deprivation are common features of the TME due to the rapid growth of tumors outpacing their blood supply.¹³⁰ As tumors grow, they often outstrip their blood supply, leading to regions with inadequate oxygen levels. This hypoxia stabilizes hypoxia-inducible factors (HIF-1α and HIF-2α), which translocate to the nucleus and induce the expression of genes promoting angiogenesis (e.g., VEGF), glycolysis, and survival.¹³¹ Cancer cells adapt to hypoxia by shifting their metabolism towards anaerobic glycolysis (the Warburg effect), increasing glucose uptake and lactate production. Additionally, hypoxia can lead to resistance to radiotherapy and some chemotherapies, as low oxygen levels reduce the generation of ROS that mediate the cytotoxic effects of these treatments. Tumor cells also face nutrient deprivation, competing for limited resources such as glucose, amino acids, and lipids due to inadequate blood supply and high metabolic demand. In response, cancer cells often upregulate autophagy to recycle cellular components and maintain homeostasis. They also exhibit metabolic flexibility, using alternative substrates like fatty acids and glutamine to sustain growth and survival. Nutrient deprivation activates stress response pathways, such as the unfolded protein response and AMPK signaling, helping cells adapt to metabolic stress.^132-134

3.3.2 Interaction with immune cells and stromal components

The interaction between cancer cells and various components of the TME, including immune cells and stromal cells, significantly influences tumor progression and therapeutic response.¹³⁵ Tumor-associated macrophages in the TME often adopt an M2-like phenotype, promoting tumor growth, angiogenesis, and immunosuppression through cytokines like IL-10 and TGF-β. Effective antitumor immune responses depend on cytotoxic T cells (CTLs), but tumors create an immunosuppressive environment that inhibits CTL function, while regulatory T cells accumulate and suppress effector T cells, aiding immune evasion.¹³⁶ Myeloid-derived suppressor cells (MDSCs) also contribute to immunosuppression by inhibiting T cell activation and promoting tumor immune evasion via arginase, nitric oxide, and ROS.¹³⁷ Cancer-associated fibroblasts (CAFs) secrete ECM components and matrix metalloproteinases (MMPs) to remodel the ECM, facilitating tumor invasion and metastasis, and produce growth factors and cytokines that promote tumor proliferation, survival, and angiogenesis. Endothelial cells respond to proangiogenic signals like VEGF from hypoxic tumor cells, forming new blood vessels that provide nutrients and oxygen to the tumor, though these vessels are often abnormal, leading to inefficient blood flow, increased permeability, and further hypoxia and nutrient deprivation. The ECM itself provides structural support and influences cell behavior through biochemical and biomechanical signals, while sequestering growth factors and cytokines, modulating their availability and activity within the TME.^{138, 139}

In summary, the TME is a dynamic and complex network that profoundly influences tumor progression and response to therapy. Hypoxia and nutrient deprivation drive metabolic adaptations in cancer cells, while interactions with immune cells and stromal components create a supportive environment for tumor growth and immune evasion. Understanding these interactions is crucial for developing strategies to target the TME and improve cancer treatment outcomes.

4.1.1 BH3 mimetics targeting Bcl-2 family proteins

Venetoclax (ABT-199) is a potent and selective inhibitor of Bcl-2, approved for treating chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) in patients with specific genetic profiles indicating Bcl-2 dependence.^{141, 158} Venetoclax has demonstrated significant efficacy in inducing apoptosis in Bcl-2-dependent cancer cells, with a favorable safety profile compared with earlier, less specific Bcl-2 inhibitors. However, cancer cells may develop resistance to BH3 mimetics by upregulating other antiapoptotic proteins like Mcl-1. To overcome resistance, BH3 mimetics are often used in combination with other therapies, such as chemotherapy, targeted therapies, or other apoptosis-inducing agents.^{158, 159}

4.1.2 Death receptor agonists

The extrinsic pathway of apoptosis is initiated by the binding of specific ligands to death receptors on the cell surface, which are part of the TNF receptor superfamily, including TRAIL receptors (DR4 and DR5) and Fas (CD95).¹⁴² Death receptor agonists are designed to activate these receptors and trigger apoptosis in cancer cells. TRAIL binds to death receptors DR4 and DR5, forming a DISC that recruits and activates caspase-8, while Fas Ligand (FasL) binds to the Fas receptor (CD95), similarly leading to DISC formation and caspase-8 activation. The activation of caspase-8 initiates a proteolytic cascade, ultimately activating effector caspases such as caspase-3, leading to apoptosis. Examples include recombinant TRAIL and agonistic antibodies targeting DR4 or DR5, which selectively induce apoptosis in cancer cells, and agonistic antibodies and recombinant FasL, which induce apoptosis in Fas-expressing cancer cells.^{143, 160} Despite promising preclinical data, clinical trials with TRAIL and FasL agonists have encountered limited efficacy in some cancers due to intrinsic or acquired resistance mechanisms. Additionally, death receptor agonists can potentially cause toxicity in normal tissues expressing death receptors, leading to adverse effects. Advances in targeted delivery systems and combination therapies are being explored to enhance the specificity and efficacy of death receptor agonists.^{144, 161}

In conclusion, apoptosis inducers such as BH3 mimetics and death receptor agonists represent promising therapeutic strategies in cancer treatment (Figure 5A). By specifically targeting the dysregulated apoptotic pathways in cancer cells, these agents aim to selectively induce cell death and improve clinical outcomes. Ongoing research and clinical trials continue to refine these approaches, addressing challenges related to resistance, specificity, and safety.

4.2.1 Inhibitors of RIPK1, RIPK3, and MLKL

To modulate necroptosis for therapeutic benefit, researchers have developed inhibitors targeting the key proteins involved in the necroptosis pathway, specifically RIPK1, RIPK3, and MLKL.¹⁴⁵ RIPK1 inhibitors block the kinase activity of RIPK1, preventing it from interacting with RIPK3 and initiating the necroptotic cascade. Examples include necrostatin-1 (Nec-1), one of the first RIPK1 inhibitors identified, which binds to the kinase domain of RIPK1, and GSK2982772, a more recent RIPK1 inhibitor currently under clinical investigation for inflammatory diseases.^{146, 163} RIPK3 inhibitors, such as GSK-872, block the kinase activity of RIPK3, preventing it from phosphorylating and activating MLKL. Targeting RIPK3 may be beneficial in diseases where RIPK3-mediated necroptosis plays a critical role, such as certain types of cancer and inflammatory conditions.^{148, 149} MLKL inhibitors, like Necrosulfonamide, prevent the oligomerization and membrane translocation of MLKL, thereby blocking the execution phase of necroptosis. Inhibition of MLKL might be useful in conditions where necroptosis contributes to pathology, such as inflammatory diseases.¹⁵⁰

4.2.2 Potential therapeutic applications and challenges

Overcoming apoptosis resistance in cancer therapy is a critical goal, and necroptosis presents a promising alternative by bypassing the resistance mechanisms that many cancers develop against apoptosis-inducing therapies.^{164, 165} Inducing necroptosis can provide an alternative route to induce cancer cell death and may enhance overall efficacy when combined with traditional therapies such as chemotherapy and radiotherapy. Additionally, necroptosis is more immunogenic than apoptosis, leading to the release of DAMPs that can stimulate an antitumor immune response. This immunogenic cell death can potentially be combined with immune checkpoint inhibitors to amplify the immune system's ability to target and destroy cancer cells. Modulating necroptosis in the TME can also alter the inflammatory milieu, potentially making it less conducive to cancer growth, and targeting necroptosis in stromal cells can disrupt supportive niches for cancer cells. However, there are significant challenges in cancer therapy, including achieving selective targeting of necroptosis in cancer cells without affecting normal cells to avoid unintended tissue damage and inflammation. Additionally, cancer cells may develop resistance mechanisms to necroptosis modulators, and inhibiting necroptosis might activate alternative cell death pathways or survival mechanisms, reducing treatment efficacy (Figure 5B). The proinflammatory nature of necroptosis, while beneficial for stimulating an immune response, could also promote tumor growth and metastasis if not carefully managed. The context-dependent effects of necroptosis in cancer, varying with tumor type, stage, and the specific TME, complicate the design of universal therapeutic strategies.^{10, 166, 167} Translating promising preclinical findings into clinical success requires rigorous clinical trials to establish the safety and efficacy of necroptosis modulators in cancer patients, and identifying patients most likely to benefit from necroptosis modulation necessitates the development and validation of biomarkers for necroptosis susceptibility.

4.3.1 Autophagy inhibitors

CQ and HCQ are lysosomotropic agents that accumulate in lysosomes, increasing the pH and inhibiting lysosomal acidification, which disrupts the fusion of autophagosomes with lysosomes and blocks the final degradation step of autophagy. Both CQ and HCQ are United States Food and Drug Administration approved for malaria and certain autoimmune diseases, and their repurposing in cancer therapy is being actively explored.¹⁵¹ In cancer therapy, these autophagy inhibitors have potential applications in enhancing chemotherapy and radiotherapy, as cancer cells often upregulate autophagy in response to these treatments as a survival mechanism. Inhibiting autophagy can sensitize cancer cells to treatment-induced death and potentially overcome resistance to targeted therapies and hormonal treatments in cancers such as breast cancer, prostate cancer, and melanoma.^168-170 However, there are challenges and considerations, including the toxicity and side effects of long-term use, such as retinal toxicity, which require careful monitoring and management in clinical settings. ¹⁷¹ Additionally, the role of autophagy in cancer is highly context dependent; in some cancers, autophagy can act as a tumor suppressor, and its inhibition might inadvertently promote tumor progression.⁷⁰

4.3.2 Dual role of autophagy in cancer therapy

Autophagy plays a dual role in cancer therapy as both a tumor suppressor and a tumor promoter. As a tumor suppressor, autophagy prevents tumorigenesis in the early stages by reducing oxidative stress and genomic instability through the removal of damaged organelles and proteins. The loss of autophagy-related genes, such as Beclin 1, has been linked to increased tumorigenesis. Additionally, autophagy can induce type II programmed cell death, or autophagic cell death, under certain conditions, aiding in the elimination of cancer cells. Conversely, as a tumor promoter, established tumors exploit autophagy to survive under stress conditions like hypoxia, nutrient deprivation, and treatment-induced stress, by providing metabolic substrates that support tumor growth and survival. This mechanism also contributes to therapeutic resistance, allowing cancer cells to adapt to and survive chemotherapy, radiotherapy, and targeted therapies.^172-174 Therapeutic strategies to target autophagy must consider its context-specific roles. Combination therapies, such as pairing HCQ with chemotherapy, can enhance treatment efficacy by preventing cancer cells from utilizing autophagy as a survival mechanism, which has shown promise in clinical trials for cancers like glioblastoma and pancreatic cancer.^{175, 176} Personalized medicine approaches, using biomarkers and genetic profiling, are essential for identifying patients who may benefit from autophagy inhibition.¹⁷⁷ Optimizing timing and dosage through adaptive therapy can maximize benefits while minimizing adverse effects, with intermittent dosing schedules potentially reducing toxicity (Figure 5C). To address resistance mechanisms, combining autophagy inhibitors with inhibitors of other survival pathways, such as the PI3K/AKT/mTOR pathway, might enhance therapeutic efficacy and prevent resistance. Continuous monitoring of autophagy activity and adaptive treatment strategies are crucial for managing resistance and improving long-term outcomes.^{178, 179}

4.4.1 Small molecules targeting GPX4

Small molecules targeting GPX4 are emerging as effective inducers of ferroptosis in cancer therapy. GPX4 plays a critical role in protecting cells from lipid peroxidation, thereby inhibiting ferroptosis.¹⁸⁰ RSL3, for instance, covalently binds to GPX4, inhibiting its activity and leading to the accumulation of LOOH, which ultimately triggers ferroptosis. RSL3 has shown efficacy in various cancer models, particularly those with high levels of ROS and lipid peroxidation, such as cancers with mutations in the Ras/Raf/MEK pathway that often exhibit elevated oxidative stress.^{154, 181} Similarly, ML210 is a potent and selective GPX4 inhibitor that disrupts the detoxification of lipid peroxides, leading to ferroptosis. Preclinical studies have demonstrated the promise of ML210, including its efficacy in models of drug-resistant cancer cells, making it a valuable tool for studying ferroptosis and its therapeutic potential.¹⁸² Additionally, iron chelators and lipid peroxidation modulators play a crucial role in ferroptosis by influencing iron levels and lipid peroxidation. Iron, through the Fenton reaction, catalyzes the formation of lipid peroxides, and modulating these processes can impact ferroptosis and its application in cancer therapy.^{183, 184}

4.4.2 Iron chelators and lipid peroxidation modulators

Iron chelators and lipid peroxidation modulators play crucial roles in ferroptosis and its therapeutic application in cancer therapy. Iron chelators like deferoxamine and deferiprone bind free iron, reducing its availability and thereby inhibiting ferroptosis. In cancer therapy, iron chelators can prevent ferroptosis in normal cells, reducing off-target effects and toxicity, and in some contexts, they can be combined with ferroptosis inducers to selectively target cancer cells with high iron content or to modulate the timing of ferroptosis induction.¹⁵⁵ Lipid peroxidation modulators can either enhance or inhibit lipid peroxide formation. For example, liproxstatin-1 is a ferroptosis inhibitor that prevents lipid peroxidation, while agents that promote lipid peroxidation can induce ferroptosis. Modulating lipid peroxidation provides a strategic approach to control ferroptosis; enhancing lipid peroxidation can synergize with GPX4 inhibitors to potentiate ferroptosis in cancer cells, whereas lipid peroxidation inhibitors can protect normal tissues, improving the safety profile of ferroptosis-based therapies.¹⁵⁴ Therapeutic strategies for ferroptosis induction include combination therapies that integrate ferroptosis inducers with other cancer treatments (e.g., chemotherapy, targeted therapy, immunotherapy) to enhance anticancer efficacy by overcoming resistance mechanisms and promoting synergistic cell death.¹⁸⁵ For instance, combining GPX4 inhibitors with agents that increase ROS levels or inhibit other antioxidant pathways can potentiate ferroptosis and enhance cancer cell killing. Targeting susceptible cancers, particularly those with high oxidative stress or specific genetic mutations (e.g., Ras-driven cancers), can maximize therapeutic benefits, with biomarker-driven approaches helping to identify patients most likely to benefit from ferroptosis-based therapies. Minimizing off-target effects involves selecting appropriate iron chelators and lipid peroxidation modulators to protect normal cells, and developing targeted delivery systems, like nanoparticles or conjugates, to improve the specificity and efficacy of ferroptosis inducers.^{186, 187} Overcoming resistance to ferroptosis, which can arise through various mechanisms such as upregulation of antioxidant pathways or alterations in iron metabolism, is crucial for the success of ferroptosis-based therapies. Combination strategies targeting multiple pathways involved in ferroptosis can help overcome resistance and improve treatment outcomes (Figure 5D).^{164, 188}

4.5.1 Activators of inflammatory caspases

Activators of inflammatory caspases, particularly caspase-1, caspase-4, caspase-5, and caspase-11, hold significant therapeutic potential in cancer therapy by inducing pyroptosis and enhancing antitumor immunity. Activators of caspase-1 can induce pyroptosis in cancer cells, leading to tumor cell death and the release of inflammatory cytokines that recruit and activate immune cells, thereby creating an immunogenic TME and potentially enhancing antitumor immunity. Activators of caspase-4/5/11 can induce pyroptosis, particularly in cancer cells resistant to apoptosis or other forms of cell death, and enhance the immunogenicity of the tumor, promoting antitumor immune responses.^189-191

4.5.2 Potential for immune activation and cancer therapy

The potential for immune activation and cancer therapy through pyroptosis induction is significant, offering promising avenues for enhancing antitumor responses. Pyroptosis leads to the release of proinflammatory cytokines such as IL-1β and IL-18, which activate and recruit immune cells, including dendritic cells, macrophages, and T cells. This cytokine milieu enhances tumor antigen presentation and promotes a robust antitumor immune response. Additionally, the release of tumor antigens and danger signals during pyroptosis can enhance the activation and maturation of dendritic cells, improving antigen presentation to T cells and boosting adaptive immunity.^{192, 193} Combining pyroptosis inducers with immune checkpoint inhibitors (e.g., anti-PD-1, anti-CTLA-4) can increase tumor immunogenicity and overcome immune suppression in the TME, enhancing the efficacy of these therapies. Likewise, pyroptosis induction can be combined with adoptive cell therapies, such as CAR-T cells or TCR-engineered T cells, to create a more favorable immune microenvironment and enhance their effectiveness.^{194, 195} Pyroptosis provides an alternative cell death pathway that can be targeted in cancers resistant to apoptosis and can turn immunologically “cold” tumors into “hot” tumors, making them more responsive to immunotherapies. To minimize off-target effects, targeted delivery systems like nanoparticle delivery or tumor-specific promoters can specifically deliver pyroptosis inducers to tumor cells, reducing systemic inflammation. Prodrug approaches can also be used to activate pyroptosis inducers specifically within the TME, mitigating the risk of systemic toxicity (Figure 5E).^{196, 197}

5.1.1 Overview of ongoing and completed trials

The clinical translation of therapies targeting cell death pathways is an active area of research, with numerous ongoing and completed clinical trials showcasing promising outcomes. Apoptosis inducers, such as BCL-2 inhibitors, have shown significant results.¹⁹⁸ Venetoclax (ABT-199), particularly in hematological malignancies like CLL and AML, has demonstrated high response rates and improved progression-free survival in CLL and encouraging responses in AML, especially in older patients.^{199, 200} Navitoclax (ABT-263), targeting BCL-2/BCL-XL, has shown efficacy in combination with standard therapies in lymphoma and small cell lung cancer but has thrombocytopenia as a dose-limiting toxicity due to BCL-XL inhibition.^{201, 202} Necroptosis inducers, including RIPK1/RIPK3 inhibitors, are also under investigation.¹⁴⁶ GSK2982772, a RIPK1 inhibitor, has been well tolerated in a Phase 1 trial with healthy volunteers, though further studies are needed to assess its efficacy in cancer.²⁰³ Nec-1, another RIPK1 inhibitor, has shown tumor growth reduction in preclinical animal models and potential for combination therapy.²⁰⁴ Pyroptosis inducers, such as caspase-1 activators, are being explored for their role in cancer therapy. VX-765, a caspase-1 inhibitor, demonstrated safety and tolerability in nononcology settings, and preclinical studies suggest its potential in cancer.²⁰⁵ Additionally, caspase-4/5/11 activators like TAK-242, a TLR4 inhibitor studied for sepsis, have shown effectiveness in reducing inflammatory responses and are being considered for repurposing in cancer therapy.^{206, 207} The key therapeutic agents targeting the cell death pathway in cancer clinical trials are shown in Table 2.

5.1.2 Key findings and clinical outcomes

Key findings and clinical outcomes of therapies targeting cell death pathways underscore their efficacy and safety. Apoptosis-inducing agents, particularly BCL-2 inhibitors like venetoclax, have shown high response rates in hematological malignancies. Combining cell death pathway modulators with existing therapies, such as chemotherapy and immunotherapy, often enhances efficacy and overcomes resistance.^{208, 209} However, managing toxicity, such as thrombocytopenia with navitoclax, is crucial, necessitating dose adjustments and supportive care.²¹⁰ Despite these advancements, challenges remain, including the development of resistance mechanisms by tumor cells, the need for selective targeting to minimize off-target effects, and the identification of predictive biomarkers for patient selection and response monitoring. Future directions focus on identifying and validating novel targets within cell death pathways, combining cell death inducers with immunotherapies, targeted therapies, and conventional treatments to enhance therapeutic outcomes, and leveraging advances in genomics and biomarker discovery to refine patient selection and treatment customization, ultimately improving clinical outcomes.^{211, 212}

5.2.1 Mechanisms of resistance to cell death-targeting therapies

Genetic mutations and varied cellular mechanisms contribute significantly to resistance against therapies targeting cell death pathways. Mutations in BCL-2 family proteins, such as BAX or BAK, can block the permeabilization of the mitochondrial outer membrane, an important step in apoptosis. Mutations in TP53, the gene responsible for p53, reduce the ability to trigger apoptosis in response to DNA damage and other cellular stress signals. Increased levels of antiapoptotic proteins such as BCL-2 and MCL-1 can bind to proapoptotic proteins, preventing apoptosis and making cells resistant to BCL-2 inhibitors like venetoclax. Likewise, a decrease in proapoptotic proteins like BAX and BAK, or the absence of BH3-only proteins like BIM, PUMA, and NOXA, disrupts the apoptotic process, leading to resistance.^216-218 Cancer cells often upregulate autophagy in response to stress, including treatment with cell death inducers, providing a survival advantage through cytoprotective autophagy.²¹⁹ Epigenetic modifications, such as DNA methylation and histone modification, can silence genes involved in apoptosis, necroptosis, and pyroptosis, further contributing to resistance.^{220, 221} Additionally, cancer cells can activate compensatory survival pathways like the PI3K/AKT/mTOR pathway or the NF-κB pathway to counteract the effects of cell death-targeting therapies.²²²

5.2.2 Strategies to overcome resistance

Combination therapies offer promising strategies to enhance therapeutic efficacy and overcome resistance in cancer treatment.^{223, 224} Targeting multiple pathways by combining cell death inducers with agents that inhibit compensatory survival pathways, such as combining BCL-2 inhibitors with PI3K inhibitors, can prevent the activation of alternative survival mechanisms. Synergistic drug combinations, like using BCL-2 inhibitors with MCL-1 inhibitors, target different components of the apoptotic pathway, effectively inducing apoptosis. Developing specific MCL-1 inhibitors and dual inhibitors that target multiple antiapoptotic proteins simultaneously can further overcome resistance by preventing cancer cells from compensating through alternative pathways.^225-227 Epigenetic modulation, using agents like DNA methylation inhibitors or histone deacetylase inhibitors, can restore the expression of proapoptotic genes and enhance cell death.^{228, 229} Inhibiting autophagy with agents such as CQ or HCQ, in combination with cell death inducers, can prevent the protective effect of autophagy and enhance cancer cell death.^{230, 231} Enhancing the immune response through immunotherapy, such as combining cell death-targeting therapies with immune checkpoint inhibitors, can help eliminate resistant cancer cells via immune-mediated mechanisms.²³² Additionally, cancer vaccines or adoptive T cell therapies targeting tumor-specific antigens released during cell death can bolster the immune response against resistant cells.²³³ Precision medicine approaches, including biomarker-guided therapy and comprehensive genomic profiling, enable the identification of resistance biomarkers and genetic alterations, guiding the selection of appropriate combination therapies and personalized treatment regimens for individual patients.²³⁴

5.3.1 Synergistic effects with chemotherapy, radiotherapy, and immunotherapy

Chemotherapy often induces cell death through DNA damage, disruption of replication, or inhibition of cellular division. When combined with cell death-targeting agents, this dual assault can enhance apoptosis. For example, BCL-2 inhibitors like Venetoclax, when combined with standard chemotherapy agents such as cytarabine, have demonstrated enhanced efficacy in AML.²⁴⁵ Similarly, PARP inhibitors, which target DNA repair mechanisms, can be combined with DNA-damaging chemotherapies to enhance cancer cell death in BRCA-mutated cancers.^{246, 247} Radiotherapy induces DNA damage and generates ROS, leading to cancer cell death. Cell death-targeting agents can sensitize cancer cells to the effects of radiation. For instance, combining radiotherapy with apoptosis-inducing agents like TRAIL receptor agonists can enhance the proapoptotic effects of radiation.²⁴⁸ Additionally, radiation can induce cytoprotective autophagy, and combining it with autophagy inhibitors can enhance the effectiveness of radiotherapy.²⁴⁹ Immunotherapies, such as immune checkpoint inhibitors (e.g., anti-PD-1/PD-L1, anti-CTLA-4), enhance the immune system's ability to recognize and destroy cancer cells. Cell death-targeting therapies can increase the release of tumor antigens, enhancing the immune response. For example, combining checkpoint inhibitors with apoptosis-inducing agents can amplify the immune-mediated killing of cancer cells, and cell death-targeting therapies combined with adoptive T cell therapies can enhance the presentation of tumor antigens, improving T cell recognition and killing of cancer cells.^250-252

5.3.2 Potential benefits and challenges

Enhanced efficacy in cancer treatment can be achieved through combination therapies, which often produce synergistic effects, making the combined treatment more effective than the sum of individual therapies and overcoming resistance by targeting multiple pathways. This approach can lead to better treatment outcomes and potentially allow for lower doses of each agent, thereby reducing overall toxicity and side effects associated with high-dose monotherapy.²⁵³ Additionally, combination therapies can target heterogeneous tumors more comprehensively by addressing different subpopulations of cancer cells within the tumor.²⁵⁴ Moreover, cell death-targeting therapies can enhance the release of tumor antigens, improving the efficacy of immunotherapies and stimulating a more robust immune response.⁵¹ However, the complexity of treatment regimens poses significant challenges, including optimizing the timing, dosing, and sequencing of therapies to maximize synergy and minimize toxicity, as well as ensuring patient compliance, particularly for those with multiple comorbidities. Increased toxicity is another concern, as combining therapies can elevate the risk of adverse effects and off-target interactions, necessitating close monitoring.²⁵⁵ Developing predictive biomarkers to identify which patients will benefit from specific combinations is crucial but challenging, requiring extensive research and validation. Tumors may also develop adaptive resistance mechanisms to combination therapies, necessitating ongoing research to counter evolving resistance patterns. Furthermore, the financial burden of combination therapies can be substantial, posing challenges to healthcare systems and patients, and ensuring equitable access to these treatments, particularly in resource-limited settings, remains a significant issue.

6.1.1 High-throughput screening and omics approaches

High-throughput screening (HTS) and omics approaches are pivotal in advancing cancer therapy.^{256, 257} HTS technology enables the rapid testing of thousands to millions of compounds or genetic perturbations, facilitating the identification of new regulators of cell death. Applications of HTS include screening small molecules, CRISPR/Cas9 libraries, or RNA interference libraries to uncover novel drug targets and signaling pathways involved in cell death. For example, screening for compounds that selectively induce cell death in cancer cells but not in normal cells can lead to the discovery of new therapeutic agents with minimal side effects.^258-260 Omics approaches, such as genomics, proteomics, and metabolomics, also play a crucial role. Genomics through whole-genome sequencing and transcriptomics can identify mutations and gene expression changes associated with cell death regulation. Proteomics, using mass spectrometry, can reveal alterations in protein expression, posttranslational modifications, and protein-protein interactions involved in cell death pathways. Metabolomics can identify changes in metabolic pathways that influence cell death, providing insights into the metabolic vulnerabilities of cancer cells. Integrating data from genomics, proteomics, and metabolomics can offer a comprehensive understanding of cell death mechanisms and identify potential therapeutic targets.^261-263

6.1.2 Novel biomarkers for therapy response

Predictive biomarkers play a crucial role in cancer therapy by guiding treatment selection and improving patient outcomes, with examples including BRCA mutations for PARP inhibitors and PD-L1 expression for immune checkpoint inhibitors, which help identify patients likely to benefit from these therapies. Prognostic biomarkers provide information about the likely course of the disease, aiding in stratifying patients based on risk and tailoring treatment intensity. For instance, biomarkers indicating high levels of apoptosis or autophagy can inform prognosis and therapeutic strategies. Dynamic biomarkers, which change in response to treatment, offer real-time feedback on therapeutic efficacy and guide adjustments in therapy. Examples include circulating tumor DNA (ctDNA) or specific protein markers in blood samples, which can be monitored to assess treatment response and detect early signs of resistance.^264-267

6.2.1 Patient-specific targeting of cell death pathways

Genetic profiling of tumors allows for the identification of mutations and alterations in cell death pathways unique to each patient, such as mutations in the TP53 gene, which plays a crucial role in apoptosis and can guide the use of therapies that exploit p53-dependent cell death mechanisms.^{268, 269} Functional genomics approaches, including CRISPR screens, can identify essential genes and pathways in individual tumors, thereby guiding the selection of targeted therapies. For example, CRISPR screens can uncover synthetic lethal interactions, revealing vulnerabilities unique to specific genetic contexts and enabling personalized therapeutic strategies.^270-272

6.2.2 Tailoring treatments based on genetic and molecular profiles

Customized drug combinations, tailored to the specific genetic and molecular profile of a patient's tumor, can enhance efficacy and reduce toxicity. For instance, combining BCL-2 inhibitors with other targeted therapies in patients with specific genetic alterations in apoptotic pathways can be particularly effective. Adaptive therapy involves adjusting treatment based on real-time monitoring of tumor response and resistance patterns, thereby tailoring therapy to the evolving tumor landscape. An example of this approach is using ctDNA to monitor tumor evolution and adjust therapy accordingly, preventing the outgrowth of resistant clones.^{273, 274}

6.3.1 Nanotechnology and targeted delivery

Nanoparticle-based delivery systems offer significant advantages by enhancing the delivery of therapeutic agents to tumor cells while minimizing off-target effects. For example, liposomal formulations of chemotherapeutic agents, such as liposomal doxorubicin, improve drug accumulation in tumors and reduce systemic toxicity.²⁷⁵ Additionally, targeted delivery systems use ligands, antibodies, or peptides to direct therapeutic agents specifically to tumor cells. An example of this is antibody–drug conjugates, which combine a monoclonal antibody specific to a tumor antigen with a cytotoxic drug, thereby enhancing selective delivery to cancer cells.^276-278

6.3.2 Overcoming barriers to effective drug delivery

The TME, characterized by hypoxia, a dense ECM, and abnormal vasculature, poses significant challenges to drug delivery. Strategies to modify the TME, such as using agents that normalize tumor blood vessels or degrade the ECM, can enhance drug penetration. For example, combining VEGF inhibitors with chemotherapeutic agents can improve vascular normalization and drug delivery. Drug resistance, due to mechanisms like efflux pumps and drug metabolism, also reduces drug efficacy. Strategies to overcome resistance include co-delivering efflux pump inhibitors or using prodrugs that are specifically activated within the tumor environment. An example is prodrugs activated by enzymes overexpressed in tumors, such as MMPs, ensuring selective activation within the tumor.^279-281

All in all, the future of cancer therapy lies in the integration of HTS, omics approaches, personalized medicine, and advanced drug delivery systems. Identifying new cell death regulators and developing novel biomarkers will enable more precise targeting of therapies. Personalized medicine approaches, tailored to the genetic and molecular profiles of individual patients, promise to enhance treatment efficacy and minimize toxicity. Advances in drug delivery systems, including nanotechnology and targeted delivery, will overcome current barriers and improve therapeutic outcomes. Continued research and innovation in these areas are essential to realize the full potential of these strategies in cancer treatment.

Despite the remarkable advancements in cancer therapy technologies, such as HTS, omics approaches, personalized medicine, and advanced drug delivery systems, significant challenges and limitations persist in their practical applications. A primary concern is the high cost and complexity associated with these technologies, which can restrict accessibility and scalability in clinical settings, particularly in low-resource environments. Additionally, the variability in individual patient responses presents a substantial challenge in effectively tailoring and predicting treatment outcomes through personalized medicine. Moreover, technological limitations in accurately modeling and predicting complex biological systems may result in unforeseen consequences and reduced efficacy of targeted therapies. Finally, regulatory and ethical issues surrounding the use of genetically tailored treatments, along with the potential for unintended off-target effects, pose critical concerns that must be addressed to ensure the safe and equitable implementation of these emerging technologies in cancer treatment. These challenges highlight the necessity for ongoing research, technological improvements, and the development of frameworks to effectively manage these issues.