The mitogen-activated protein kinase (MAPK) network encompasses the kinases RAF/MEK/ERK. The association of extracellular ligands to cell surface ERBB receptors activates this kinase pathway (Yarden and Sliwkowski, 2001). Upon binding to ligand, the cytoplasmic region of the receptor shifts and permits binding of the Guanine exchange factor (GEF) Son of Sevenless (SOS). This GEF promotes the exchange of GDP for GTP in the RAS binding pocket (Chardin et al., 1993). The GTP bound RAS has an increased affinity for RAF kinases, which are recruited to the plasma membrane. This re-localisation induces phosphorylation of the N-terminal auto-inhibitory region and promotes dimerization, with both events being required for full kinase activity (Mason et al., 1999; Rajakulendran et al., 2009). This initiates the sequential phosphorylation of MEK and ERK (Figure 1). The final component of this pathway, ERK, phosphorylates many targets with a wide range of cellular functions, including regulation of transcription factors (von Kriegsheim et al., 2009).

Overexpression of ERBB family members and mutation of RAS/RAF are commonly observed events in cancer. For example, ERBB2 (HER2) amplification is observed in up to 30% of breast cancers and is closely associated with poor prognosis and relapse (Slamon et al., 1987). This led to the development of a HER2 targeted monoclonal antibody, trastuzumab, which prevents ligand association with HER2 and ablates pathway activity, significantly improving patient survival (Slamon et al., 2001; Agus et al., 2002). This reliance of tumours upon ERBB signalling is also observed in lung cancer with particularly high prevalence of ERBB1 (EGFR) mutations (Zhang et al., 2016). Therefore, the current frontline therapy for advanced non-small cell lung cancer (NSCLC) are receptor tyrosine kinase inhibitors. In addition to the importance of EGFR at late stages of disease, the activity of ERBB receptors is critical during the early stages of KRAS-driven lung tumours, with the addition of tyrosine kinase inhibitors to MEK inhibition improving survival of a KRas driven murine lung cancer model (Kruspig et al., 2018; Moll et al., 2018). The mutation of Ras and RAF is commonly observed in all cancers (Prior et al., 2020; Owsley et al., 2021). Therefore, new wave drug development efforts are directly focusing on these targets. The development of inhibitors which specifically recognise mutant forms of RAS improves tumour specificity and potency (Hansen et al., 2018; Lou et al., 2019; Punekar et al., 2022). Inhibitors against MEK and BRAF are being developed and utilised, particularly in the context of melanoma (Hauschild et al., 2012; Caunt et al., 2015). The impact of pathway inhibition upon cancer is demonstrative of the critical role ERBB signalling plays in maintaining the adapted disease state.

The plasma membrane forms a critical barrier between the extracellular space and the intracellular milieu. This membrane is formed from a complex structured lipid bilayer, which houses numerous proteins fundamental to regulation of signalling and import/export dynamics. While these proteins are key to transferring signals from the extracellular space into the cell, the lipids themselves also have fundamental roles in cellular signalling. One group of lipids with crucial signalling activity are the phosphatidylinositols (PI), which can be phosphorylated at two positions converting PI to phosphatidylinositol-4,5-bisphosphate (PIP₂). The two consecutive phosphorylation events are mediated through the phosphatidylinositol 4-kinases (PI4Ks), the PtdInsP-5-OH kinases (PIP(5)Ks), and the Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) (Whitman et al., 1987; Rameh et al., 1997; Rameh and Blind, 2023). These kinases are grouped into class II and class III. Class I refers to kinases which can targets the 3’ site and encompasses PI3K, which is responsible for phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP₂) yielding phosphatidylinositol-3,4,5-trisphosphate (PIP₃) (Toker and Cantley, 1997). This process of phosphorylation is not monodirectional with antagonism of this process being driven by phosphatase and tension homolog deleted on chromosome 10 (PTEN), which converts PIP₃ into PIP₂ (Maehama and Dixon, 1998; Myers et al., 1998; Stambolic et al., 1998). The abundance of PIP₃ determines the activity of two kinases - 3-phosphoinositide-dependent protein kinase-1 (PDK1) and Protein kinase B (PKB/AKT). In the presence of high PIP₃ concentration both AKT and PDK1 are recruited to the plasma membrane, through their pleckstrin homology (PH1) domains, where AKT undergoes a structural shift following association with PIP₃ increasing catalytic activity of PDK1 towards T308 AKT (Alessi et al., 1997; Currie et al., 1999). In parallel, to support complete activation of AKT kinase activity mammalian target of rapamycin 2 (MTORC2) phosphorylates AKT S473 (Sarbassov et al., 2005). The downstream targets of AKT are numerous and overlap significantly with other members of the AGC kinase family. However, one critical target of AKT is glycogen synthase kinase-3 (GSK3) (Figure 2). GSK3 was initially described as kinase which inhibits the activity of glycogen synthase, but its functions now stretch far beyond (Embi et al., 1980). For example, it is critically involved in the regulation of cellular ROS responses through targeting of nuclear factor erythroid 2–related factor 2 (NRF2) for degradation (Rada et al., 2011).

In addition to generation of PIP₃, PIP₂ can also be hydrolysed by phospholipase C to form IP₃ and diacylglycerol (DAG). The IP₃ subsequently translocates to the endoplasmic reticulum and stimulates calcium ion (Ca²⁺) release through binding to IP3 Receptors (IP3R) (Michell and Allan, 1975; Streb et al., 1983). In addition, DAG stimulates activity of proteins containing C1 domains, such as protein kinase C (PKC) and protein kinase D (PKD) (Takai et al., 1979a; Takai et al., 1979b; Van Lint et al., 1995; Matthews et al., 1997).

The intrinsic link between PI3K/AKT/GSK3 signalling and cell growth makes it a key oncogenic driver pathway, previously reviewed in depth (Manning and Cantley, 2007; Hoxhaj and Manning, 2020). This growing evidence has further supported the initial characterisation of PTEN as a critical cellular tumour suppressor. Attempts have been made to suppress the overactivity of this pathway in cancer. However, the development of PI3K inhibitors has been hampered by the toxicity associated with candidate compounds (Ellis and Ma, 2019). While inhibition of AKT has been trialled multiple times with low toxicity. However, has shown poor clinical efficacy as a monotherapy (Hua et al., 2021).

Recognition of cellular energy perturbations is required to support cellular viability. The fundamental molecules of the intracellular energy system are AMP, ADP, and ATP. Therefore, multiple pathways have evolved to monitor levels are these molecules. One such pathway is centred on AMPK, which is a heterotrimeric complex comprising a catalytic subunit, α, and two regulatory subunits, β and γ. The kinase activity of this complex is stimulated by binding of AMP and ADP which induces a structural change permitting phosphorylation of T172 by Liver kinase B1 (LKB1) to fully activate kinase activity (Figure 3) (Yeh et al., 1980; Carling et al., 1987; Oakhill et al., 2011; Xiao et al., 2011). Activated AMPK inhibits pathways such as gluconeogenesis and translation, while promoting catalytic processes including increased glucose utilisation and mobilisation of lipid stores. The combined activity of these regulatory cascades supports the restoration of cellular ATP generation. A comprehensive overview of the field’s history can be found in these two reviews (Hardie et al., 2011; Mihaylova and Shaw, 2011). Importantly, AMPK is the archetypical member of the AMPK related kinase (ARK) family, which consists of 12 other members all of which are activated by LKB1 mediated phosphorylation at a T-loop threonine (Lizcano et al., 2004). An overview of the functions of the kinases within this family can be found in these two reviews (Bright et al., 2009; Monteverde et al., 2015). One of the kinases most closely related to AMPK is NUAK1, which has a diverse set of functions including regulation of centrosome number, MYC-dependent splicing control, HIPPO signalling control, and importantly cellular ROS responses (Humbert et al., 2010; Port et al., 2018; Cossa et al., 2021; Whyte et al., 2023). The phosphorylation of MYPT at S445 by NUAK1 increases the activity of GSK3β for NRF2 and suppresses the cellular ROS response thereby reducing the capacity of cancer cells to maintain adaptation to high ROS production (Port et al., 2018). In addition, the over-expression of NUAK1 in the cytoplasm supports glycolytic switch of cancer cells (Escalona et al., 2020). As demonstrated by LKB1/AMPK activation following increased AMP levels, these nucleotides have critical signalling capacity. A further signalling role for these nucleotide molecules is through the adenylyl cyclases (AC). There are ten AC found within the human cell, nine of which are membrane bound and one which is cytoplasmic. These generate cAMP by conversion of ATP following the activation of G-protein coupled receptors (GPCRs) (Scholich et al., 1999; Pierce et al., 2002). Importantly, the generation of cAMP is spatially regulated, generating microdomains of signalling (Zaccolo and Pozzan, 2002; Pierre et al., 2009). The increased cAMP activates protein kinase A (PKA), which phosphorylates a multitude of downstream targets (Figure 3) (Corbin and Krebs, 1969).

Given the roles of AMPK signalling in regulation of energy homeostasis this pathway has major roles underpinning cancer cell viability (Hardie, 2013). A recent example demonstrates the importance of AMPK during prostate cancer development through control of catabolic signalling (Penfold et al., 2023). Following previous work which found increased sensitivity of LKB1 deficient lung cancers due to the rewiring of AMPK signalling (Shackelford et al., 2013). The functional importance of the ARK family for cancer persistence is not limited to AMPK. The loss of NUAK1 expression induces apoptosis in cancer cells with over-expression of MYC, a commonly observed phenotype of tumorigenesis (Liu et al., 2012).

The regulation of cellular calcium influx is most closely associated with neuronal cell function and transcriptional responses, with the major family of import channels referred to as voltage gated Ca²⁺ channels (VGCC) (Hardingham et al., 1997; Catterall, 2011). However, the regulation of cellular calcium in non-neuronal cell types is equally critical (Berridge et al., 2003; Berridge, 2012; Bootman and Bultynck, 2020). One pathway of calcium signalling is driven through the calcium/calmodulin associated kinases (CaMK) and phosphatases. The calcium binding protein calmodulin is a key component of this pathway, containing 4 EF-hand domains capable of binding calcium and driving association with binding partners (Means and Dedman, 1980; Babu et al., 1985). These binding partners include the CaMK kinases which has two isoforms α (CAMKKα) and β (CAMKKβ), or through binding to CAMK kinase 2 (CAMKK2). These kinases phosphorylate a threonine residue on the activation loop of calcium/calmodulin kinases (CaMK) and permit full catalytic activation in combination with binding to calmodulin (Figure 4). An overview of the early discoveries and regulatory steps in these pathways was recently published (Marcelo et al., 2016). Interestingly, in addition to regulation of CAMKI and CAMKIV, CAMKK2 was also found to activate AMPK in certain tissues or following loss of LKB1 (Woods et al., 2005; Green et al., 2011; Mairet-Coello et al., 2013).

The perturbation of calcium signalling is associated with the onset of various neurodegenerative diseases (Mattson, 2007). However, the involvement of the downstream kinases is understudied by comparison. The loss of CAMKK2 expression has been associated with altered transferrin regulation which can drive over production of ROS (Sabbir, 2018). The loss of CAMKK2 activity also protects against the synaptoxicity of Amyloid β in murine in vitro (Thornton et al., 2011; Mairet-Coello et al., 2013). In the context of cancer CAMKK2 has been associated with development of prostate cancer, activation of AKT signalling in ovarian cancer, and regulation of immune microenvironment in breast cancer (Gocher et al., 2017; Penfold et al., 2018; Racioppi et al., 2019).

The mitochondrial network is highly dynamic and, in response to various intracellular and extracellular signalling events, can change morphological state. Mitochondria are either fused into an elongated continuous network or undergo fission to compartmentalise mitochondria into punctate units. The proteins that regulate this process can be split into two groups: the pro-fission proteins and pro-fusion proteins. The phosphorylation of proteins involved in the processes of mitochondrial fusion and fission has major consequences for protein function. However, do these phosphorylation events have the capacity to support the development of disease states and is it a necessary step during cellular adaptation?

The three major regulators of mitochondrial fission are Dynamin related protein 1 (DRP1), fission protein 1 (FIS1), and mitochondrial fission/fusion factor (MFF). When mitochondrial fission is initiated DRP1 is recruited to the mitochondrial membrane bound receptors MFF and FIS1 (James et al., 2003; Stojanovski et al., 2004; Gandre-Babbe and van der Bliek, 2008; Otera et al., 2010). A DRP1 oligomer encircles the mitochondria and through dynamin activity cleaves through the mitochondrial membrane (Mears et al., 2011; Kalia et al., 2018). The phosphorylation of these proteins has a major impact upon activity and will define mitochondrial fission/fusion status in multiple scenarios. The importance of DRP1 serine 616 (S616) and serine 637 (S637) for controlling the activity of DRP1 during cell cycle, cell death, and mitophagy induction will now be covered. In general, phosphorylation of DRP1 S616 is associated with increased mitochondrial fission and phosphorylation of S637 is associated with fusion. During mitosis, DRP1 associates with the RALA/RALBP1 complex and is phosphorylated at S616 by the CDK1/cyclin B complex, supporting fission of the mitochondrial network (Kashatus et al., 2011). Loss of DRP1 expression or over-expression of a non-phosphorylatable form suppressed this mitochondrial fragmentation and antagonised progression through mitosis (Taguchi et al., 2007). Following the stimulation of cells with growth factors, the phosphorylation of DRP1 at S616 by ERK drives mitochondrial fission, which can be blocked by the application of MAPK inhibitors (Kashatus et al., 2015; Serasinghe et al., 2015). The morphological state of the mitochondrial network will influence the capacity of cells to induce apoptosis. In cardiac cells the chronic activation of β-adrenergic receptors (β-AR) over-activates CaMKKII which directly phosphorylates DRP1 at S616. The increased phosphorylation supports induction of cell death and cardiac damage, which can be reversed by the application of CAMKKII inhibitors (Xu et al., 2016). In contrast, the phosphorylation of DRP1 at S637 by PKA drives mitochondrial fusion and increases resistance to cell death following treatment with apoptosis inducers. This phenotype can be reverted by overexpression of a non-phosphorylatable DRP1 S637A mutant (Chang and Blackstone, 2007; Cribbs and Strack, 2007). Interestingly, the increased expression of dominant negative mutant of Rab32 T39N can increase phosphorylation of S637 through regulation of PKA recruitment to mitochondria (Bui et al., 2010). The induction of mitophagy is an important process which permits clearance of unwanted mitochondria. This requires encapsulation of mitochondria by auto-phagosome membranes (Palikaras et al., 2018). The phosphorylation of DRP1 at S616 by PINK1 kinase drives mitochondrial fission and supports mitophagy (Han et al., 2020). Phosphorylation events with defined phosphorylation sites mediating mitochondrial morphology are summarised in Table 1. The stability of phosphorylation events is defined by a balance between the activity of kinases and phosphatases. The phosphatase Calcineurin dephosphorylates the S637 site of DRP1 and regulates its translocation to mitochondria inducing mitochondrial fusion (Cereghetti et al., 2008; Sandebring et al., 2009).

In addition to the phosphorylation of S616 and S637 multiple alternative sites also define DRP1 function. Phosphorylation of DRP1 by CaMKIα at S600 increases the translocation to mitochondria, mediated by an increased affinity for FIS1 (Han et al., 2008). The phosphorylation of DRP1 at S693 by GSK3β can drive resistance to H₂O₂ induced fragmentation of the mitochondrial network and apoptosis induction, which is further recapitulated by overexpression of S693D mutant DRP1 (Chou et al., 2012). In addition to phosphorylation at S693, GSK3β also phosphorylates S40 and S44 sites in neuronal cells, this can drive increased GTPase activity and increase fragmentation of the mitochondria, this is also driven by phospho-mimetic mutants at S40 and S44 (Yan et al., 2015).

The majority of investigations have focused upon DRP1 phosphorylation events in isolation. However, the interplay between these modifications poses an intriguing question. For example, the phosphorylation of S600 enhances phosphorylation at S579, with both of these sites required for complete mitochondrial fragmentation. Interestingly, loss of S600 induces disrupted mitochondrial morphology and metabolic capacity in mice carrying a S600A mutation in DRP1 (Valera-Alberni et al., 2021). This study suggests that further investigation of the interplay between phosphorylation sites is critical to our complete understanding of mitochondrial morphology regulators.

These studies demonstrate the nuanced regulatory system that controls DRP1 activity which is cell type and cell state specific. A further complicating factor is the parallel phosphorylation of other factors which control mitochondrial fission/fusion dynamics. Much like DRP1 both FIS1 and MFF are regulated by phosphorylation events. FIS1 phosphorylation at Y38 by MET drives mitochondrial fission through increased recruitment of DRP1 to the mitochondrial outer membrane in hepatocellular carcinoma cell lines (Qi et al., 2016). MFF phosphorylation at S129 and S146 by AMPK and drives mitochondrial fission responses (Ducommun et al., 2015; Toyama et al., 2016). Protein kinase D (PKD) targets MFF S155, S172, and S275, with loss of phosphorylation at these sites driving defects in mitochondrial fission and leads to abnormal chromosome segregation during mitosis (Figure 5) (Pangou et al., 2021).

The antagonists to mitochondrial fission are the proteins which control mitochondrial fusion. The process of mitochondrial fusion requires proteins to act at both the outer and inner mitochondrial membranes. The outer membrane proteins are Mitofusin 1 (MFN1) and Mitofusin 2 (MFN2) which drive fusion of adjacent mitochondrial membranes through GTPase activity (Ishihara et al., 2004; Qi et al., 2016; Li et al., 2019). Both proteins are subject to multiple phosphorylation events which impact their functional activity. Phosphorylation of MFN1 by ERK at T562 reduces mitochondrial fusion and instead promotes BAK association with the mitochondrial membrane more efficiently, inducing apoptosis (Pyakurel et al., 2015). MFN2 is phosphorylated by PINK1 at T111 and S442 and triggers parkin association with the mitochondria inducing mitophagy (Chen and Dorn, 2013; Li et al., 2022). The dephosphorylation of MFN2 S27 by Mitochondrial Serine/Threonine protein phosphatase (PGAM5) protects MFN2 from degradation (Nag et al., 2023). In contrast, the JNK dependent phosphorylation of MFN2 recruits ubiquitination complexes which degrade MFN2 and increase the cellular sensitivity to apoptosis (Figure 5) (Leboucher et al., 2012).

The regulation of mitochondrial inner membrane fusion is controlled by the cleavage of OPA1 (Mishra et al., 2014). While phosphorylation moieties have been detected on OPA-1 no upstream kinases have been characterised. The cleavage of OPA-1 is regulated by OMA-1. Phosphorylation of OMA-1 by GSK3β has been detected and is critical during oocyte development (Nishi and Lin, 2005; Shirayama et al., 2006; Stitzel et al., 2006).

The perturbed function of fission and fusion proteins underpins the development of many diseases. For example, DRP1 function is critical for the development of RAS driven skin, pancreas, brain, squamous cell carcinoma, and colorectal cancers, with loss of expression preventing cellular transformation (Serasinghe et al., 2015; Xie et al., 2015; Kitamura et al., 2017; Nagdas et al., 2019; Sessions et al., 2022; Zhu et al., 2022). Interestingly, the development of lung adenocarcinoma seems independent of DRP1 but is instead dependent upon the expression of OPA1 (Sessions et al., 2022). Two recent reviews provide a further exploration of fusion/fission protein function in disease (Serasinghe and Chipuk, 2017; Yapa et al., 2021). However, how does the alteration to specific phosphorylation sites, rather than total protein function, drive disease?

Many studies have focused on the phosphorylation of DRP1 and its role in tumorigenesis. Interestingly the S616 and S637 sites seem to act as a regulatory convergence point for the kinase pathways outlined earlier. Firstly, the phosphorylation of DRP1 at S616 is required for the transformation of melanocytes cells which overexpress RAF. However, even in the absence of RAF the over-expression of constitutively phosphorylated DRP1 is sufficient to support transformation of melanocytes (Serasinghe et al., 2015). This provided strong evidence of DRP1's capacity to drive oncogenic transformation of cells. This is further supported by multiple reports of ERK mediated DRP1 S616 and S637 phosphorylation driving cellular transformation. For example, S616 phosphorylation supports mitochondrial functionality in lung cancer and promotes proliferation and migration of tumour cells (Chung et al., 2021). While the phosphorylation of DRP1 at S637 by ERK is required for the generation of inducible-pluripotent stem cells in tumours (Prieto et al., 2016). Interestingly, the nucleotide dependent kinase pathways also target the S616 and S637 sites. Salt inducible kinase 2 (SIK2) phosphorylates DRP1 S616 in ovarian cancer cells and therefore promotes mitochondrial fission, supporting tumour cell survival (Han et al., 2020). The PKA mediated phosphorylation of DRP1 S637 is required for hepatocellular carcinoma cell response to energy stress. This was driven by elongation of the mitochondrial network, increased cristae formation, and a switch towards oxidative phosphorylation, this could be reversed by overexpression of DRP1 S637A mutants (Li et al., 2017).

In addition to these early events in tumorigenesis, the modulation of S616 phosphorylation has also been associated with adaptation of tumours towards cancer therapies. Firstly, mitochondrial dynamics are associated with the resistance of tumour cells to chemotherapy. The induction of DRP1 mediated mitochondrial fragmentation and glycolytic switch in T cell acute lymphoblastic leukaemia co-cultured with mesenchymal stem cells drives chemoresistance (Cai et al., 2016). The activation of ERK signalling through HMGB1 and activation of RAGE pathway induced phosphorylation of DRP1 S616 and drove autophagy increasing chemoresistance of colorectal cell lines (Huang et al., 2018). Secondly, the exposure of tumours to ionising radiation (IR) is a common front-line treatment. However, the impact of IR on the mitochondria is poorly understood. One study identified CAMKKII as driving phosphorylation of DRP1 S616, which supports network fragmentation in response to IR (Bo et al., 2018). Finally, the treatment with hormonal therapy is common for cancers, such as, breast and ovarian. The resistance of tumours to these treatments is a key problem, and the cellular mechanisms which drive this process need further investigation. Interestingly, tamoxifen resistant breast cancer cell lines showed a decreased respiratory capacity, increased superoxide, and mitochondrial fission with increased DRP1 S637 phosphorylation (Tomkova et al., 2019). The identification of increased S637 in fragmented mitochondrial networks was intriguing, as this site is usually associated with fusion. Therefore, more work will be required to establish the kinase pathway which drives this phosphorylation and if this necessary for tamoxifen resistance.

The phosphorylation of DRP1 has been studied in multiple contexts and supports the adaptation of cells during the development of disease, such as cancer. However, the regulation of other fusion and fission family members by PTMs is greatly understudied and the potential for this process to underpin disease development is significant. This is demonstrated by the PINK1 mediated phosphorylation of MFN2 S442 which promotes its degradation and supports proliferation of lung cancer cell lines (Dasgupta et al., 2021).

The evidence for mitochondrial fusion and fission protein phosphorylation supporting disease has driven research to pharmacologically target these events in disease. Despite the well-characterised role of mitochondrial fusion and fission proteins in cancer development and progression no clinical trials have explored targeting mitochondrial morphology as a viable anti-cancer therapeutic strategy. Moreover, clinical and preclinical trials exploring kinase inhibition in advanced models of cancer, for example, ERK and MEK inhibition, did not characterise downstream effects on mitochondria morphology and function (Caunt et al., 2015; Moschos et al., 2018). However, clinical intervention targeting mitochondrial kinases has been investigated in neurodegeneration and cardiac ischemia. The inhibition of ERK restores the balance between mitochondrial fusion and fission to repair aberrant mitochondrial morphology in hybrid neurons with incorporated platelet mitochondria from Alzheimer’s disease patients highlighting the promise behind ERK targeting (Gan et al., 2014). Importantly, the astragaloside IV derivative LS-102 alleviated myocardial ischemia reperfusion injury in H9c2 cells by blocking GSK3β-mediated DRP1 phosphorylation at S616 and hence mitochondrial fragmentation in a similar manner to the GSK3β inhibitor AR-A014418 (Chen et al., 2020).

The clinical benefits of targeting mitochondrial morphology can be further illustrated by trials in cardiovascular disease. A phase II clinical study investigates Sovateltide (PMZ-1620; IRL-1620) for the treatment of hypoxic-ischemic encephalopathy (HIE) (NCT05514340). Sovateltide is an endothelin B receptor (ETBR) agonist promoting higher differentiation of neuronal progenitor cells with improved mitochondrial morphology and thus function (Ranjan et al., 2020). The above investigation follows from Sovateltide’s first approval in May 2023 in India for the treatment of cerebral ischemic stroke within 24 h of stroke onset (Keam, 2023). Evidence of Trimetazidine cardioprotective effects via improved balance between mitochondrial fusion and fission underpinned a phase II clinical trial investigating Trimetazidine’s effects on patients with pulmonary hypertension (PAH) (Shi et al., 2017) (NCT02102672). The investigators had hypothesised that fatty acid oxidation inhibition by Trimetazidine will restore mitochondrial morphology and function in PAH patients. In general, preclinical therapeutic strategies aimed at supporting healthy mitochondrial networks including DRP1 inhibition and MFN activation were deemed central to cardioprotection (Hernandez-Resendiz et al., 2023). These data point to the potential behind kinase targeting to modulate mitochondrial networks in diseases presenting with aberrant mitochondrial morphology.

Nevertheless, the number of clinical trials targeting mitochondrial morphology control both through kinase regulation and directly is still low highlighting an unmet need.

BAD binds to BCL-2 family members, thereby preventing these proteins from associating with BAX/BAK (Yang et al., 1995). The capacity of BAD to bind with BCL-2 family members is regulated by phosphorylation. The phosphorylation of S112 and S136 is mediated by AKT in response to growth factor availability and suppresses apoptosis induction (del Peso et al., 1997; Datta et al., 1997). The phosphorylation of these two sites in combination with a further phosphorylation at S155 promotes association with 14-3-3 proteins and degradation of BAD (Datta et al., 2000). The S112 site was found to be particularly promiscuous, with ribosomal s6 kinase (RSK), PKA, and Raf-1 also capable of targeting this site (Bonni et al., 1999; Harada et al., 1999; Polzien et al., 2009). Further to these previously identified sites, the phosphorylation of S111 by PAK1 has also been identified, as with the S112 site this supports association with 14-3-3 proteins and promotes cell survival through availability of BCL-2 proteins (Polzien et al., 2009; Ye et al., 2011).

BID is a BH3-only protein which is cleaved by caspase activity following activation of extrinsic apoptosis signals (Li et al., 1998; Luo et al., 1998). Cleaved BID, known as tBID, subsequently translocates to the mitochondrial outer membrane and induces BAX/BAK activity to drive MOMP (Gross et al., 1999; Eskes et al., 2000; Wei et al., 2000). The phosphorylation of BID at S61 and S64 by casein kinase I (CKI) and casein kinase II (CKII) suppress apoptosis induction by FAS ligand, through reduced cleavage of Bid by caspase 8 (Desagher et al., 2001; Degli Esposti et al., 2003). In contrast, a study identified phosphorylation of BID by JNK at T59 prevents caspase 8 cleavage, but despite the lack of cleavage this drives apoptosis (Prakasam et al., 2014). The interaction between the different phospho-sites identified in these studies and the context specificity of this impact requires further elucidation. In addition to extrinsic apoptosis signalling, BID is a mediator of intrinsic apoptosis. Following DNA-damage during S-phase BID is phosphorylated at S61 by the DNA damage kinase ATM and functions to maintain the IR-induced cell cycle checkpoint (Kamer et al., 2005; Zinkel et al., 2005). In addition, increased BID S66 phosphorylation is required to induce apoptosis of cells stalled at the mitotic checkpoint, with a non-phosphorylatable form of BID suppressing cell death following mitotic arrest (Wang et al., 2014).

BIM is a component of the intrinsic apoptosis cascade which competes for binding of pro-survival BCL-2 family members (Kim et al., 2006). There are multiple isoforms of BIM which all compete with BAX and BAK for binding to BCL-2 family proteins. Initially three isoforms were identified BIM_EL, BIM_L, and BIM_s (O’Connor et al., 1998). However, further work has identified a further isoform, BimAD, which shares capacity to induce apoptosis (Marani et al., 2002). The expression and stability of BIM is a key determinant of cell apoptotic potential, as demonstrated by the genetic requirement for BIM during MYC-induced apoptosis in multiple tissues (Muthalagu et al., 2014). Multiple target residues within BIM are phosphorylated to regulate stability. For example, in response to nerve growth factor (NGF), phosphorylation of BIM at S109 and T110 was detected in a MEK dependent manner, protecting neurons from BIM-induced cell death (Biswas and Greene, 2002). The phosphorylation of BIM by ERK1/2 at S65 and S69 following growth factor stimulation destabilising the protein and suppresses apoptosis (Ley et al., 2003; Luciano et al., 2003; Ley et al., 2004; Ley et al., 2005b). The activity of ERK mediated phosphorylation of BIM is also required to promote cell survival following detachment of cells (Fukazawa et al., 2004). Phosphorylation at S44, T56, and S58 by JNK drives apoptosis by activating BIM (Lei and Davis, 2003). A second study also found JNK mediated phosphorylation of BIM by JNK drives activation of mitochondrial driven mitosis in neurons. However, this study identified S65 as the target site (Putcha et al., 2003). The phosphorylation of BIM by both ERK and JNK suggests a complex interplay between the two kinases (Ley et al., 2005a). Finally, in a B cell line dependent upon interleukin-3 (IL-3) survival is determined by phosphorylation of BIM. In this cell line the phosphorylation of S87 by AKT is sufficient to drive ubiquitination and promote survival (Qi et al., 2006).

As with other members of the cell death induction pathway BIM has also been identified to have roles regulating mitosis dependent apoptosis. Aurora A has been identified to phosphorylate BIM at S93, S94, and S98 and drives degradation, this downregulates apoptotic signals through mitosis and supports successful completion of cell cycle (Moustafa-Kamal et al., 2013). The phosphorylation in a MEK dependent pathway was also required to successfully progress through mitosis (Graos et al., 2005).

Both PUMA and NOXA are members of the BCL-2 family protein family that are expressed following p53 activation, for instance in response to DNA damage (Oda et al., 2000; Han et al., 2001; Nakano and Vousden, 2001; Yu et al., 2001). Both proteins are crucial mediators of p53 induced cell death (Villunger et al., 2003). The phosphorylation of NOXA by CDK5 at S13 is critical for suppressing its apoptotic function when glucose is available. This was confirmed by knockdown and ectopic expression of S12A NOXA mutants which increased apoptosis induction (Lowman et al., 2010; Karim et al., 2015). Interestingly PUMA interacts with HER2 and is phosphorylated at Y152 and Y172 this phosphorylation increased degradation of the protein and reduces apoptosis induction (Carpenter et al., 2013).

BAX and BAK form pores in the outer mitochondrial membrane and drive mitochondrial permeabilization (Wei et al., 2001; Pena-Blanco and Garcia-Saez, 2018). While the activity of these proteins is primarily controlled by their release from pro-survival BCL-2 complexes, phosphorylation can also modify their activity at the mitochondrial membrane. The phosphorylation of BAX by GSK3β at S163 reduced both localisation to the mitochondria and pore formation, a finding which could be recapitulated by over-expression of a S163A BAX mutant (Linseman et al., 2004; Arokium et al., 2007). The initial mechanism identified for BAK phospho-regulation was regulation by non-receptor tyrosine phosphatases (PTPNs). The phosphate groups at Y108 and S117 must be removed to support the full activation of BAK apoptotic activity (Fox et al., 2010; Azad et al., 2012). More recently, the kinase responsible for the phosphorylation of these tyrosine residues has been identified as the protein kinase BMX (Fox and Storey, 2015).

As previously discussed, the pro-survival proteins - BCL-2, BCL-XL, MCL-1, and BCL-W act to antagonise cell death induction by binding to BAX and BAK. The phosphorylation of BCL-2 by JNK at T56, S70, T74, and S87 provided a critical link between mitogenic signalling and propensity of cells to induce apoptosis (Maundrell et al., 1997). The importance of S87 for BCL-2 stability and activity was further demonstrated as mutation of S87A drives degradation of the protein (Breitschopf et al., 2000). P38 MAPK drives phosphorylation of the S87 and T56 sites (De Chiara et al., 2006). Finally, PKCα can phosphorylate BCL-2 S70 and this increases the resistance to apoptosis (Ruvolo et al., 1998). A study tried to reconcile the specific impact of each phosphorylation event upon the induction of apoptosis. The development of this model permitted prediction of apoptosis induction based on the modelling of BCL-2 phosphorylation status (Song et al., 2019). The phosphorylation of MCL-1 by ERK at T92 and T163 is required to prevent degradation of MCL-1 and thereby promote cell survival (Ding et al., 2008). The production of H₂O₂, is a carefully balanced cellular signalling event, and over-production can lead to significant cellular toxicity and apoptosis induction. The identification of JNK dependent phosphorylation of S121 and Thr163 of MCL-1 in conditions of high H₂O₂ was instrumental in linking high ROS to apoptosis induction (Inoshita et al., 2002). Interestingly, the phosphorylation of MCL-1 S159 and T163 is also controlled by dephosphorylation through PP2A (Slomp et al., 2021).

Phosphorylation events involved in mediating the function of pro- and anti-apoptotic proteins and thus cellular apoptosis are summarised in Table 2.

The development of diseases, such as cancer, is often reliant upon the avoidance of apoptosis, with one key mechanism of disease adaptation involving the modulation of BH3 family expression levels (Certo et al., 2006). A second key adaptative mechanism in disease, is altered regulation of apoptotic protein post-translation modification. For example, the phosphorylation of BAD S118 and S99 has been linked to altered activity of mitochondrial electron transport chain utilisation, which supports tumour growth (Mann et al., 2019). Therefore, understanding the kinase pathways responsible for these changes provides novel treatment targets and elucidates treatment resistance mechanisms. The MAPK signalling pathway targets multiple sites which support the development of cancer. The overexpression of BRAF decreases anoikis induced cell death in melanoma cells through increased phosphorylation of BAD S75 (Boisvert-Adamo and Aplin, 2008). Additionally, the overexpression of B-RAF-V600E drove increased proliferation of cells in a BAD dependent manner (Polzien et al., 2011). In melanoma, BAD is phosphorylated through MEK/ERK dependent signalling, supporting cell survival, this is an adaptation from melanocytes where these phosphorylation’s are mainly driven by the RSK signalling pathway (Eisenmann et al., 2003). The adaptation of MAPK signalling also supports the development of blood cancers. A common mutation during the development of acute myeloid leukaemia (AML) is the FLT3-ITD mutation (Nakao et al., 1996; Griffith et al., 2004). In this setting, the activation of ERK/MEK pathway drives anti-apoptotic phosphorylation of BAD and suppresses cell death (Watanabe et al., 2019). Further, the overexpression of PIM3, a commonly observed oncogene in AML, utilises these mechanisms to suppress tumour cell death (Luo et al., 2020). The regulation of cell death proteins by PIM3 can also be observed in pancreatic cancer. Whereby, the PIM3 mediated phosphorylation of BAD is constitutive and is vital for prevention of apoptosis (Li et al., 2006). The stem cell niche is also supported by the phosphorylation of BIM in response to mitogenic signalling through the ERK pathway (Harada et al., 2004). Finally, the modulation of the MAPK family kinases in breast cancer has multiple impacts upon tumour survival. Over expression of HER2 is a common adaptation found in breast cancer cells. The direct phosphorylation of PUMA by HER2 provides a mechanism by which this initial adaptation can drive cell survival and suppression of apoptosis (Carpenter et al., 2013). MCL-1 phosphorylation by ERK is supported by PIN1 and supports survival of breast cancer cells through stabilisation of MCL-1 (Ding et al., 2008). The increased activity of the ERK signalling pathway in mammary epithelial cells also drives phosphorylation of BIM and prevents apoptosis induction and supports avoidance of anoikis which promotes metastasis (Marani et al., 2004; Collins et al., 2005). The overexpression of BAG-1 promotes phosphorylation at RAF driven BAD sites and inhibits apoptosis in breast cancer (Kizilboga et al., 2019).

The lipid secondary messenger kinase pathways support the adaptation of cell death pathways in cancer. For example, multiple members of the PKC family can drive phosphorylation of BAD. In prostate cancer cells PKCε can drive phosphorylation of S112 and promotes cell survival (Meshki et al., 2010). In parallel, G-protein coupled oestrogen receptor 1 (GPER), can drive increased activity of PKA signalling which increases BAD phosphorylation and increases breast cancer cell survival (Chan et al., 2020). In both Glioma and lung cancer, PKC iota phosphorylates S112 and promotes cell survival (Jin et al., 2005; Desai et al., 2011). BAD expressed in neuronal cells can be phosphorylated by PINK1 at S112 and can promote survival in these cell types (Wan et al., 2018). Increased AKT activation and phosphorylation of BAD is associated with the adaptation of the breast cancer cell line MCF-7Ca cells to oestrogen deprivation (Sabnis et al., 2005).

The interplay between these pathways has been assessed in melanocytes, suggesting inhibition of adapted ERK/PKA/PKC pathways simultaneously yields the highest apoptosis induction (Sastry et al., 2020). This phenotype can also be seen in immortalised epidermal keratinocytes which are dependent upon BIM to prevent anoikis (Quadros et al., 2006).

The DNA damage kinase cascade has also been demonstrated to support modification of cell death related proteins. The phosphorylation of BID by ATM is critical for supporting the hematopoietic stem cell niche and is therefore a prime target for kinase driven adaptation in disease development (Maryanovich et al., 2012). This is supported by the increased development time to leukemogenesis in ATM −/− mice is BID expression is also suppressed, suggesting BID is required to prevent apoptosis of leukemic cells (Biswas et al., 2013).

This underlying adaptation of kinase signalling in tumours can have impacts beyond tumour initiation. This can also define the resistance to chemotherapeutic intervention. For instance, MAPK pathway activation can induce BIM dependent resistance to paclitaxel in epithelial tumours (Tan et al., 2005). The phosphorylation of BAK by BMX increases the resistance of cancer cells to chemotherapy, suggesting this adaptation supports the disease persistence (Fox and Storey, 2015). The development of resistance to tyrosine kinase inhibitors is a typical adaptation in treated non-small cell lung cancer. This is mechanistically supported by the phosphorylation of MCL-1 which stabilises the protein and increases expression levels (Wu et al., 2016). Resistance of ovarian cancer to chemotherapies can be closely tied to phosphorylation of BAD (Marchion et al., 2011). The phosphorylation of key residues in the apoptotic proteins is modulated by a balance between kinases and phosphatases. Therefore, it is unsurprising that modulation of PP2A activity regulates the dephosphorylation of S112 and mediates the sensitivity of chronic lymphocytic leukaemia (CLL) cells to cAMP pathway inhibition (Moon and Lerner, 2003).

Cellular apoptosis has a dual role in disease, with cancers usually resisting cell death by apoptosis and neuronal apoptosis implicated in various neurological diseases. BAD phosphorylation is perhaps the most characterised apoptosis-related phosphorylation event that has demonstrated promise in the cancer therapeutic landscape. Treatment with the PI3K inhibitor Piticlisib (GDC-0941) leads to BAD dephosphorylation at S112 and S136 sensitising glioblastoma cells to the BCL-2 inhibitor ABT-263 (Pareja et al., 2014). A study investigating novel treatments for peritoneal carcinomatosis, discovered that a combination of mitomycin C (a DNA crosslinker) and the mTOR inhibitor rapamycin successfully induced apoptosis by BAD dephosphorylation by inactivating p70 S6 ribosomal kinase (S6K1) (Song et al., 2014). These examples serve as proof of concept that targeting phosphorylation events implicated in apoptosis could be a promising anti-cancer approach. Additionally, combination therapies as discussed in the context of Debio 1143 + PD-1/PD-L1 checkpoint blockade and Piticlisib + ABT-263 suggest a possibility for kinase targeting to serve as a sensitisation strategy rather than a single-agent therapeutic in anti-cancer regimens. Dexmedetomidine (DEX) is a commonly used sedation medication that exerts its action via agonism of the central pre- and postsynaptic α2-receptors in the locus coeruleus (Weerink et al., 2017). Interestingly, preclinical efforts have shown that DEX could reduce brain damage after ischemia-reperfusion by decreasing ER stress-induced neuronal apoptosis (Zhai et al., 2019). DEX treatment also reduced phosphorylated-JNK suggesting in this context JNK may have pro-apoptotic properties considering its role of a double-edged sword in apoptosis regulation. This evidence of kinase activity modulation as a downstream effect of pharmacological interventions mediating cellular apoptosis highlights the promise behind direct kinase targeting which is currently a considerably under-researched area. DEX’s translational potential was investigated in an interventional clinical trial hoping to show neuroprotective effects of DEX administration during brain surgery by means of reduced apoptosis and subsequent neuronal injury (NCT02878707).

Current clinical attempts in the cancer therapeutic landscape aim to induce apoptosis in a selective manner through multiple mechanisms. Debio 1143 (AT-406, SM-406) is a second mitochondrial-derived activator of caspases (SMAC) mimetic and acts as antagonist of the inhibitor of apoptosis proteins (IAPs) aiming to restore apoptosis in cancer cells (Cai et al., 2011). A different approach to inducing apoptosis is the inhibition of BCL-2 to prevent it from opposing pro-apoptotic protein function. A phase I clinical trial is investigating the BCL-2 inhibitor TQB3909 for the treatment of patients with relapsed or refractory advanced malignant tumours (NCT04975204). The variety of pharmacological efforts to restore apoptosis in cancer cells confirms the need to explore diverse upstream of apoptosis signalling events (incl. phosphorylation) to combat the heterogenous nature of cancer.

One of the first reports of phosphorylation mediated regulation of mitochondrial calcium overexpressed multiple PKC isoforms. Each PKC isoform had a differential impact upon cellular calcium stores, with PKCζ increasing histamine mediated calcium flux into mitochondria (Pinton et al., 2004). While no phospho-target was determined for this activity it suggested the importance of post-translational modification for mitochondrial calcium regulation. VDAC complexes form pores on the outer mitochondrial membrane responsible for the uptake of Ca²⁺. In addition to this role, some reports have implicated a role for VDAC in MOMP (Kim et al., 2019). Importantly, the phosphorylation of VDAC can both inhibit and promote MOMP. While the phosphorylation of VDAC1 by NEK1 at S193 protects cells from induction of mitochondrial membrane permeabilization (Chen et al., 2009; Chen et al., 2010). The phosphorylation of VDAC1 by GSK3β at S12 and S103 increases sensitivity to endostatin induced death by increasing VDAC1 expression levels and supporting mPTP opening and caspase 3 activation (Yuan et al., 2008). The mechanisms by which phosphorylation mediates this effect is incompletely understood. However, one key element is loss of hexokinase II (HK2) binding to VDAC1, which can act to protect against cell death in response to chemotherapy in cancer cells (Pastorino et al., 2005). It is also possible that the effect is mediated by the effectiveness of MCU to permit mitochondrial calcium entry. The identification of a tyrosine phosphorylation event on MCU driven by PYK2 activity resulting in MCU oligomerisation suggests an acute phosphorylation-driven method of regulating mitochondrial calcium (O-Uchi et al., 2014). This is further supported by the AMPK mediated phosphorylation of MCU, which drives a mitotic specific calcium influx to drive increased ATP production, with loss of this regulation leading to spindle assembly delay (Zhao et al., 2019). Finally, PKC mediates phosphorylation of S92, while not functionally characterised, is hypothesised to alter the dimerization capacity of MCU tetramers (Lee et al., 2020). This functional activation of MCU is predominantly regulated by the complex subunits MICU1 and MICU2 (Kamer et al., 2017; Wang et al., 2020). The phosphorylation of MICU1 by AKT at S124 increases mitochondrial calcium (Marchi et al., 2019). The NA(+)/Ca(2+) exchanger (NCLX) is a major route of calcium efflux from the mitochondria into the cytoplasm (Palty et al., 2010). The phosphorylation of NCLX by PKA can rescue the typical calcium overload observed in PINK1 deficient cells, this mechanism is critical for maintenance of neuronal cell viability (Kostic et al., 2015; Rozenfeld et al., 2022).

A major finding was the identification of a promyelocytic leukaemia (PML) body at MAMs, which associated with IP3R, AKT, and PP2A. Furthermore, this AKT/PP2A mediated regulation of IP3R was critical to the regulation of calcium release (Zaccolo and Pozzan, 2002). This supported previous work which suggested the phosphorylation of IP3R by AKT at S2681 reduced the efflux of calcium from the ER and protected cells from apoptosis (Marchi et al., 2008; Szado et al., 2008). AKT is also the kinase responsible for phosphorylation of Phosphofurin acidic cluster sorting protein 2 (PACS-2) at S437, which increases its association with 14-3-3 proteins preventing its role in apoptosis. This association can be reversed by the addition of TRAIL to cells which induces dephosphorylation and promotes apoptosis (Aslan et al., 2009). This cascade was further elucidated by the finding that MTORC2 is responsible for the activation of AKT in these conditions (Betz et al., 2013). In addition to these phosphorylation dynamics during apoptosis regulation, the control of these processes has been observed in a cell cycle dependent manner. The modification of IP3R1 by CDK1/cyclin B complex at T799 drives increased IP3 binding and Ca²⁺ release, suggesting a cell cycle dependency (Malathi et al., 2005). Finally, altered calcium signalling is a key element of cellular differentiation. The phosphorylation of IP3R Y353 by Fyn kinase drives prolonged calcium release from the ER during B cell activation, linking this protein with developmental Ca²⁺ release (Cui et al., 2004; deSouza et al., 2007).

The modification of MAM structural proteins is not limited to IP3R and VDAC components. The interaction between VAPB-PTPIP51 is impacted by GSK3β with the inhibition of kinase activity increasing the complex formation (Stoica et al., 2014). In addition, PTPIP51 was also found to be phosphorylated at T176 by c-Src. The altered phosphorylation of this site drove differential interaction with 14-3-3 family proteins and PKA (Brobeil et al., 2012). Given this interaction with 14-3-3 proteins, this could impact cellular localisation of PTPIP51. The impact of this phosphorylation site upon the activity of PTPIP51 in MAM control has not been investigated.

Evidently both Ca²⁺ homeostasis and MAMs are tightly regulated by phosphorylation with all discussed phosphorylation events summarised in Table 3.

The importance of mitochondrial Ca²⁺ regulation and MAMs makes them common points of adaptation in disease. While mitochondrial lipid homeostasis, mitochondrial Ca²⁺ and metabolism are regulated by MAM proteins, the investigation of how these pathways drive disease is still developing. Some promising work suggests a role for regulation of these processes by kinase signalling. One study has shown interaction of RIPK1 with MCU increases mitochondrial Ca²⁺ level and promotes proliferation of colorectal cancer cells (Zeng et al., 2018). The importance of Ca²⁺ in cancer is further supported by the AKT mediated phosphorylation of MICU1 S124 promoting proliferation of three different oncogenic cell lines (Marchi et al., 2019). Finally, the dysregulation of mitochondrial function is increasingly associated with the onset of neurological diseases. The regulation of VAPB-PTPIP51 in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) by FUS metabolism defects through GSK3β mediated regulation of complex formation and supports disease development (Stoica et al., 2016).

There is an intricate relationship between MAMs and mitochondrial Ca²⁺ homeostasis. Hence it is not surprising that therapeutic approaches aimed at stabilising the mitochondria-ER assembly have demonstrated promise in the treatment of neurological diseases driven by defective MAMs and dysregulated Ca²⁺ signalling. While current clinical trials have not targeted the kinases which regulate these proteins, the ongoing studies show the potential promise of such an approach.

The Sigma-1 receptor (Sig-1R) is a chaperone at the ER (in MAMs) that maintains Ca²⁺ homeostasis and MAMs stability among others. Sig-1R is sensitive to Ca²⁺ concentrations and mediates Ca²⁺ signalling through IP3R. Sig-1R deficiency or absence negatively impacts mitochondria-ER contacts and Ca²⁺ homeostasis inducing neuron degeneration and other symptoms of neurological/neurodegenerative disorders. Therefore, Sig-1R agonists including Pridopidine (ACR16) presented as a promising therapeutic opportunity. A phase 3, randomised, double-blind, placebo-controlled trial investigated the effect of Pridopidine in Huntington’s disease (HD) patients (de Yebenes et al., 2011) (NCT00665223, MermaiHD). While no change in the modified motor score was observed at 26 weeks, the authors suggested that Pridopidine may influence the motor phenotype in HD. A phase 2/3 study, (NCT04615923), part of HEALEY ALS (NCT04297683), investigated the use of Pridopidine as a potential treatment of amyotrophic lateral sclerosis (ALS). While the study did not meet its primary endpoint at 24 weeks–positive change in ALS Functional Rating Score-Revised (ALSFRS-R), ALS patients on Pridopidine presented with improved speech measures.

There is evidence of successful preclinical targeting of MCU to inhibit Ca²⁺ exchange between the ER and mitochondria in models of Parkinson’s disease (Soman et al., 2017; Liu et al., 2020). Additionally, the mitochondrial MAM protein cyclophilin D is vital for MAM integrity and interactions required for efficient insulin signalling (Tubbs et al., 2014). These data suggested modulating MAMs and mitochondrial Ca²⁺ homeostasis to be a viable therapeutic strategy. Further work is required to investigate the potential of kinase targeting in this context.

The cellular response to high ROS levels partly mediated by transcriptional response which increase expression of detoxifying enzymes. The Nuclear respiratory factor (NRF) family consists of two members, NRF1 and NRF2. Both proteins are key regulators of nuclear localised mitochondrial genes. In particular, the oxidation of the cysteines in nuclear factor-erythroid factor 2-related factor 2 (NRF2) binding partner Kelch-like ECH-associated protein 1 (KEAP1), induces NRF2 dissociation and permits nuclear translocation activating multiple signalling cascades that support the cellular ROS response (Itoh et al., 1999; Sekhar et al., 2002; McMahon et al., 2003; Stewart et al., 2003). In addition to this dissociation with KEAP1, NRF2 nuclear translocation is also regulated by phosphorylation. GSK3β phosphorylates NRF2, preventing nuclear accumulation of the latter and this activity is countered by inhibitory phosphorylation of GSK3β on Ser9 by AKT (Table 4) (Rojo et al., 2008). Further complexity to this cascade emerged with the discovery that the AMPK-related kinase NUAK1 limits the rate of myosin phosphatase targeting subunit 1 (MYPT1)/PP1-dependent dephosphorylation of GSK3β Ser9 (Figure 8) (Port et al., 2018). The phosphorylation of S445, S472, and S910 by NUAK1 increases association of MYPT1 with PP1, inhibiting phosphatase activity. This prevents the dephosphorylation of GSK3β S9 and inhibits kinase activity. This prevents the dephosphorylation of GSK3β S9 and inhibits kinase activity. The suppression of GSK3β reduces the phosphorylation of NRF2 at canonical sites in the phospho-degron, S334 and S338, decreasing association with SCF/β-TrCP and subsequent degradation (Rada et al., 2011). This increases the pool of NRF2 available to induce signalling responses. Interestingly, in addition to the roles in redox control, NRF2 association with PGAM5 and KEAP1 was found to regulate mitochondrial motility, with the loss of either NRF2 or PGAM5 from this complex driving MIRO1 degradation and mitochondrial clustering (O'Mealey et al., 2017).

A second crucial ROS response pathway are the forkhead factors of the O class (FOXOs). All members of the FOXO family have conserved target sites which, when phosphorylated by AKT, increase association with 14-3-3 family members and traffic these proteins out of the nucleus. In proliferating cells this activity of AKT inhibits nuclear translocation of FOXO factors and prevents quiescence induction (Brunet et al., 1999; Kops et al., 1999; Rena et al., 1999). Given the importance of AKT for cellular ROS response multiple mechanisms have evolved to enable FOXO activity in the presence of increased AKT kinase activity. For example, a key mechanism of regulation is the deacetylation of FOXO3 by SIRT1 in conditions of oxidative stress increasing the expression of FOXO target genes (Brunet et al., 2004). In addition, multiple kinase pathways can promote increased activity of FOXO transcriptional activity. A kinase particularly associated with both mitochondrial function and ROS is AMPK, which plays a key role in control of FOXO factors. The AMPK-mediated phosphorylation of S22 decreases binding of FOXO1 to 14-3-3 proteins, increasing nuclear localisation, while the phosphorylation of S383 and T649 increase transcription of FOXO1 target genes, such as, mSOD (Yun et al., 2014; Saline et al., 2019). During nutrient deprivation, AMPK phosphorylates FOXO3 at six sites, but of note are S413 and S588. The presence of phosphorylation at these sites increases transcriptional activity of FOXO3 and drives expression of mitochondrial genes, such as, uncoupling protein 2 (UCP2) and PPARG coactivator 1 α (PGC1α) (Greer et al., 2007). This can also directly drive oxidative stress induced expression of SODs in quiescent cells and protect against apoptosis induction (Kops et al., 2002). Importantly the cross talk between transcriptional pathways can have significant impact of ROS response outcome. The activation of FOXO3a cascade can repress MYC-driven mitochondrial gene transcriptional programs, reducing mitochondrial function, and suppressing ROS production (Delpuech et al., 2007; Ferber et al., 2012). These mechanisms suppress apoptosis in response to oxidative stress. In contrast, the phosphorylation of FOXO3 at S207 by oxidation activated mammalian Ste20-like kinases (MST1) suppresses association with 14-3-3 proteins and promotes nuclear translocation, inducing cell death (Table 4) (Lehtinen et al., 2006).

The onset of disease is associated with altered cellular response to ROS. Tumours driven by MYC, KRAS^G12D, and BRAF^V600E all show increased basal NRF2 transcriptional activity promoting tumour survival (DeNicola et al., 2011). The altered regulation of NRF2 regulation has been observed through multiple mechanisms. In BRCA1 mutant breast cancer overactive Pi3K/AKT signalling in releases GSK3β mediated suppression of NRF2 nuclear translocation, supporting tumour survival (Gorrini et al., 2014), while in colorectal cancer, the regulation of GSK3β activity by the NUAK1/MYPT/PP1 complex maintains nuclear localisation of nuclear NRF2 (Port et al., 2018). In addition, common loss of KEAP1 function in non-small cell lung cancer promotes over activity of NRF2 transcriptional cascades (Hast et al., 2014; Berger et al., 2016). In addition to supporting tumour cell survival this adapted ROS response can lead to treatment resistance (Scalera et al., 2022).

In addition to cancer, the dysregulation of ROS regulatory cascades is strongly associated with development and progression of neurodegenerative diseases (Barnham et al., 2004; Singh et al., 2019). The lipid membranes of neuronal cells are rich in poly unsaturated fatty acids (PUFAs). This lipid species is particularly sensitive to oxidative damage. The accumulation of α-synuclein (α-syn) increases ROS generation and damages lipid membranes leading to ferroptosis and neuronal cell death, with this mechanism contributing to the onset of Parkinson’s disease (Angelova et al., 2015). In a murine model of Alzheimer’s disease, increased lipid peroxidation was found to correlate strongly with subsequent deposition of amyloid β plaques, suggesting a contribution of oxidation to progression of the disease (Praticò et al., 2001).

The combination of elevated ROS and decreased endogenous antioxidants in cancer, neurodegenerative and cardiovascular diseases drove clinical and preclinical efforts to mitigate these phenotypes.

MIT-001 is a ROS scavenger that mediates graft-versus host disease via decreased high-mobility group box 1 (HMGB1) expression–an inflammatory marker and lowered oxidative stress (Im et al., 2015). An active phase II clinical trial is investigating the efficacy of three different MIT-001 doses as a preventative for oral mucositis (OM) in patients with head and neck squamous cell carcinoma HNSCC who are undergoing chemoradiotherapy (NCT04651634). Chemoradiotherapy-associated OM is usually caused by injury to basal epithelial cells inducing increased ROS levels. MIT-001 is expected to decrease ROS levels and excessive inflammation caused by the high ROS thus preventing the development of OM.

Nicotinamide riboside (NR)—a dietary supplement, is a nicotinamide adenine dinucleotide (NAD+) precursor showing promising therapeutic action via elevating the levels of NAD+ in the body (Bieganowski and Brenner, 2004). NAD+ is a coenzyme essential for numerous oxidation-reduction reactions in cells. Various NAD+ -depleting enzymes are known to be activated by oxidative stress thus exogenously elevating NAD+ levels presented as a promising antioxidant strategy in disease (Zhang et al., 2019). Several preclinical efforts supported this showing that NAD+ enhancing strategies improved outcomes in metabolic disorders (Cantó et al., 2012), neurodegenerative diseases (Schöndorf et al., 2018) and age-associated complications (Mills et al., 2016). An early phase I clinical trial is underway investigating whether NR supplementation can improve cardiac function in patients with heart failure (HF) (NCT04528004). A focus of the study will be to determine how increased blood and myocardial NAD+ levels affect mitochondrial function in HR.

Mitoquinone (MitoQ) comprises of Triphenylalkylphosphonium ion (TPP+) conjugated to the endogenous antioxidant Ubiquinone and is one of the best described mitochondria-targeted antioxidants (Kelso et al., 2001). MitoQ has been shown to reduce oxidative damage and display anti-apoptotic properties in various disease models further reiterating the complex relationship between mitochondria-driven processes (Duarte et al., 2023). MitoQ supplementation was investigated as a prevention strategy in breast cancer patients undergoing treatment with doxorubicin-adjuvant chemotherapy (NCT05146843). An expected outcome was reduction in doxorubicin-induced oxidative stress.

Interestingly, mediating oxidative stress is perhaps one of a few disease presentations with completed or ongoing clinical trials investigating dietary supplements (both NR and MitoQ among others) as the therapeutic strategy.

These studies in combination with the mechanistic evidence that GSK3β phosphorylation supports the antioxidant response machinery in cells suggest that targeting phosphorylation events associated with ROS decrease or antioxidant increase could be a viable therapeutic strategy.