ABSTRACT _FUS1/TUSC2_ (_FUS_ion_1_/TUmor _S_uppressor _C_andidate _2_) is a tumor suppressor gene (TSG) originally described as a member of the TSG cluster from human 3p21.3 chromosomal

region frequently deleted in lung cancer. Its role as a TSG in lung, breast, bone, and other cancers was demonstrated by several groups, but molecular mechanisms of its activities are

starting to unveil lately. They suggest that Fus1-dependent mechanisms are relevant in etiologies of diseases beyond cancer, such as chronic inflammation, bacterial and viral infections,

premature aging, and geriatric diseases. Here, we revisit the discovery of _FUS1_ gene in the context of tumor initiation and progression, and review 20 years of research into FUS1 functions

tumor suppressor, anti-inflammatory and anti-aging activities. SIMILAR CONTENT BEING VIEWED BY OTHERS DEREGULATION OF MITOCHONDRIAL GENE EXPRESSION IN CANCER: MECHANISMS AND THERAPEUTIC

OPPORTUNITIES Article 14 August 2024 ING2 TUMOR SUPPRESSIVE PROTEIN TRANSLOCATES INTO MITOCHONDRIA AND IS INVOLVED IN CELLULAR METABOLISM HOMEOSTASIS Article 20 May 2021 SIRT7: A NOVEL

MOLECULAR TARGET FOR PERSONALIZED CANCER TREATMENT? Article Open access 21 February 2024 INTRODUCTION Execution of cellular programs including proliferation, differentiation, apoptosis,

senescence is based on fine-tuned signal transduction cascades. Growth receptor signaling (such as receptor tyrosine kinases or RTK) should be tightly controlled at the spatial and temporal

levels to execute appropriate cell decisions. Uncontrolled RTK signaling (via EGFR, HER2/neu, or VEGFR) could be oncogenic and conducive to a variety of other pathologies [1, 2]. Indeed,

tumor suppressors integrate various signaling networks pivotal for tissue homeostasis [1, 2]. They control DNA repair (e.g., BRCA1, MSH2), cell cycle progression (RB1, CDKN2A, TGFB1),

angiogenesis (VEGF, ANGPTL4), apoptosis (TP53, BCL2), and cell adhesion (CADM1, FAT, CDH) [1,2,3,4,5,6,7,8]. A separate class of tumor suppressors is miRNAs, small hairpin RNAs regulating

gene expression (miR-7, miR-29, miR-145) [5, 6, 9] and https://bioinfo.uth.edu/TSGene/. Loss of TSG functions in malignant cells occurs at the DNA, transcriptional and post-transcriptional

levels and may be achieved via different mechanisms. According to the Knudson’s two-hit paradigm (stemming from clinical observations on _Rb_ deficiency), first hit comes from the loss of

heterozygosity (LOH) due to deletion or loss-of-function mutation in one of two TSG alleles that can be inherited as a recessive mutation. Second hit causes a biallelic mutation eliminating

the function of the remaining TSG allele. This transforms the cell towards malignancy. The effect of haploinsufficiency, when mutation of a single gene copy is sufficient for malignant

transformation, has been described for some TSGs (CDKN1B, TP53, NF1, PTEN) [7, 8]. Later, importance of epigenetic silencing via promoter hypermethylation or histone modification was also

recognized [7, 8]. At the protein level, TSG products can be functionally inactivated via proteasomal degradation (MDM2/p53 axis) or through altered cellular compartmentalization

(mislocalization of SMAD4 in cytosol vs. nucleus) [5]. Transcriptional silencing of TSGs in tumors has also been reported (TWIST/CDH1 repression axis) [5]. In this review, tracing the

history of the tumor suppressor _Fus1/Tusc2_ studies “from bench to bedside”, we discuss what is known of its role in tumor development, inflammation, and aging. Initially characterized as

one of several genes belonging to potential 3p21.3 TSG cluster [10,11,12], recently FUS1 gene enters clinical trials as a gene-based therapy in the form of plasmid DNA encapsulated in cation

lipid nanopartciles (REQORSA) in combination with other drugs for patients with non-small cell lung cancer (NSCLC) [13]. Reported ability of Fus1/Tusc2 to inhibit receptor (EGFR, PDGFR,

c-Kit) and non-receptor (c-Abl, Akt) tyrosine kinases as well as promote apoptosis [14] makes Fus1/Tusc2 a promising adjuvant for successful anti-tumor chemotherapy

(https://clinicaltrials.gov/ct2/show/NCT01455389, https://clinicaltrials.gov/ct2/show/NCT04486833). By affecting the listed above upstream pathways, Fus1/Tusc2 can potentailly prevent cancer

cell evolution towards drug-resistant variants. During the last years, ongoing revolution in immunotherapy including series of Abs targeting immune checkpoint (IC) molecules altered

therapeutic landscape in tumor treatment and significantly improved therapeutic outcomes [15,16,17]. However, broad spectrum of immune evasion mechanisms let cancer cells escape from immune

surveillance stimulated by using IC inhibitors in clinics (e.g., expression of alternative IC molecules such as TIM-3 after anti-PD-1 therapy) [18, 19]. This issue was suggested to be solved

by using REQORSA as Fus1/Tusc2-based genetic therapy in mouse model of lung cancer reduced expression of array of IC molecules (TIM-3, CTLA-4, PD-1) concomitant with anti-tumor response

[20]. Noteworthy, studies on mice with the Fus1 loss extended its biological activity beyond tumorigenesis to aging, inflammation, infections, and geriartic conditions [21,22,23]. Fus1 was

demonstrated to be the novel regulator of mitochondrial Ca2+ (miCa2+) transport [24, 25, 23]. Fine-tuning of Ca2+ signaling by Fus1/Tusc2 may control cellular senescence and its disturbance

may contribute to various pathologies. _FUS1/TUSC2_ GENE DISCOVERY AND FUNCTION The pioneering concept describing the importance of loss of chromosomal segments responsible for tumor

suppression was put forward by Theodor Boveri in 1914 [26]. In the 1980s, chromosomal abnormalities identified in small cell lung carcinoma (SCLC) demonstrated that SCLC cell lines and tumor

biopsies shared the deletion of 3p14-23 region [27, 28]. Further, this chromosomal segment was narrowed down to 3p [21,22,23, 29] and 3p21 [30] as two most common overlapping deletions in

SCLC and other lung cancers. In parallel, it was established that the 3p deletions followed the LOH pattern and matched the Knudson’s two-hit paradigm [31, 32]. The localization of TSGs in

the 3p21 region was reinforced by detection of deletions in this area in other solid (e.g., breast, cervix, and renal carcinoma) and hematopoietic (chronic myeloid leukemia) tumors

[33,34,35,36,37,38,39,40,41]. Few research groups used a microcell fusion technique to transfer 3d chromosome into tumor cells. Such hybrid tumor cells displayed signs of senescence, growth

arrest, and decreased tumorigenicity in athymic nude mice. This demonstrated a tumor suppressor potential for the entire chromosome 3 [42] and regions 3p21 [43] and 3p21.3 [44]. To perform

detailed mapping of the 3p21 region, initial analysis of growth suppression of human-mouse tumor hybrid cells was done and resulted in the isolation of a subclone HA [3] BB9F carrying a 2-Mb

fragment of human 3p21-22 chromosome with tumor suppressor function [43]. Further, this region was narrowed down to the 3p21.2-21.3 area using specific chromosomal markers [45]. A

long-range physical map spanning about 1.8-Mb DNA over the deleted region helped to construct a 700-kb clone. This library (23 cosmids and one PI phage) included a genetically defined 370-kb

segment containing lung cancer TSGs from the 3p21.3 fragment [40]. This led to the identification of a 220 kb segment deleted in primary breast cancer. By overlapping chromosomal fragments

deleted in SCLC cells, the TSGs area was narrowed down to the minimal 120 kb deletion nested within three small homozygous deletions in the 3p21.3 segment (3p21.3 C or LUCA, 3p21.3 or CER1,

and 3p21.3 T or AP20). Within this 120 kb deletion were identified eight genes: _HYAL2_, _FUS1(TUSC2)_, _RASSF1_, _BLU/ZMYND10_, _NPR2L_, _101F6_, _PL6_, and _CACNA2D2_ [11]. The original

name of _TUSC2_, _FUS1_, was introduced based on its position at the junction (FUSion) of cosmids LUCA12 and LUCA13. _FUS1_ remains to be commonly used even though it is sometimes confused

with an unrelated gene, _FUS_ [10]. Shortly after gene identification, it was demonstrated that overexpression of the _FUS1/TUSC2_ transgene in _FUS1/TUSC2_-deficient lung cancer cells

suppressed proliferation, blocked G1/S or G2/M transition, and increased doubling time implying a tumor suppressor role for FUS1/TUSC2 [46, 47]. Likewise, intratumoral adenoviral delivery of

the _Fus1/Tusc2_ transgene suppressed tumor growth and lung metastases in mice [46]. For further insights into the biological role of FUS1, _Fus1/Tusc2__−/−_ mice were developed. These

animals showed increased frequencies of lupus-like autoimmune conditions (vasculitis, glomerulonephritis, anemia, circulating autoantibodies) and spontaneous vascular tumors [22], confirming

tumor suppressor properties of Fus1. Moreover, these mice exhibited increased susceptibility to irradiation [48, 49], enhanced response to _A. baumanii_ infection [21], premature aging

[23], hearing loss [50], and olfactory and spatial memory impairments [51]. These pleiotropic effects of Fus1/Tusc2 loss expanded its role beyond tumor suppression activities. Also, these

data suggested that Fus1/Tusc2-dependent therapeutic approaches could alleviate/treat different diseases associated with mitochondrial dysfunction, which is linked to inflammation,

infection, metabolic imbalance, and aging. _FUS1/TUSC2_ LOCALIZATION, MUTATIONS, AND EXPRESSION IN NORMAL AND TUMOR TISSUES The _FUS1/TUSC2_ gene is located in the centromeric segment of

3p21.3 (chromosome 9 in mice) encoding a small (110 aa) protein without recognizable domains [10, 12]. The _TUSC2/FUS1_ gene is 3.3 kb long and contains three exons, which encode a 1691 bp

mRNA with 5’UTR (untranslated region) spanning positions 1–147 and 3’UTR spanning positions 481–169 [10] (Fig. 1A). At the beginning of 3p21.3 cluster analysis, it was expected that TSGs

from this region would possess cancer-associated spectrum mutations. Surprisingly, extensive studies found little or no mutations in the genes from 3p21.3 cluster, and in the _FUS1/TUSC2_

gene, except for a few lung cancer cell lines with gene deletions and stop mutations. Overall, the mutational rate in this area did not exceed 5% [10]. The identified stop mutation (28 bp

deletion at the 3’ terminus of the _FUS1/TUSC2_ exon 2) resulted in the expression of nonfunctional C-terminal truncation at position 82 [47]. In normal tissues_, FUS1/TUSC2_ is ubiquitously

expressed with the highest levels in all brain regions, followed by blood vessels, stomach, esophagus, colon, adrenal, pituitary, and thyroid glands, skeletal muscle, kidney, spleen, lung,

testis, and fallopian tubes (https://gtexportal.org/home/gene/TUSC2). _FUS1/TUSC2_ mRNA was detected in lung and sarcoma cancer cell lines and sarcoma tissues, but no protein expression was

detected in SCLC or non-small cell lung carcinoma (NSCLC) cells [2, 47, 52, 53]. Low or no protein expression was explained by its reduced half-life in tumor cells due to the loss of

myristoylation, a post-translational modification that prevents FUS1/TUSC2 from proteasome-mediated degradation [53]. Detailed information on systemic, cellular and molecular manifestations

of Fus1 decrease/loss/increase in normal and tumor tissues is presented in Table 1. _FUS1/TUSC2_ GENE REGULATION IN NORMAL AND TUMOR TISSUES Besides the post-translational modification

affecting _FUS1/TUSC2_ expression due to the loss of myristoylation, its expression can also be regulated at the translational level. At the 5’UTR, the _FUS1/TUSC2_ mRNA contains two

alternative highly conserved open reading frames (uORF1 and uORF2) and a secondary structure, which can suppress ribosomal scanning during translation. The 3’UTR also displays a negative

regulatory activity mediated via miRNAs and regulation of mRNA stability [54]. Thus, miR-93, miR-98, and miR-197 target 3’UTR and down-regulate expression of _FUS1/TUSC2_ mRNA (Fig. 1A,

Table 2). Elevated miR-93 and miR-197 expression correlated with reduced _FUS1/TUSC2_ expression in NSCLC tumors [55]. Other miRNAs suppressing _FUS1/TUSC2_ mRNA expression include miR-663

in ovarian cancer [56], miR-19a in lung cancer [57], miR-378 in mesenchymal stem cells [58], and miR-584 in thyroid cancer [59]. In triple-negative breast cancer cells, miR-138 binds to the

5′-UTR site on _FUS1/TUSC2_ mRNA containing translation initiation region and interferes with its translation [60]. An additional layer regulating _FUS1/TUSC2_ mRNA expression includes two

_FUS1/TUSC2_ pseudogenes (TUSC2P) on chromosomes X and Y. Their RNAs have the region identical to the 3’UTR region of _FUS1/TUSC2_ mRNA. _Tusc2P_ mRNA is complimentary to several miRNAs:

miR-17, miR-93, miR-299-3p, miR-520a, miR-608, and miR-661 (Fig. 1A). Their binding to _FUS1/TUSC2_ RNA sequesters miRNAs from interacting with the 3’UTR of _FUS1/TUSC2_ mRNA. As a result,

_FUS1/TUSC2_ mRNA expression is increased, leading to inhibited cell proliferation, survival, migration, invasion, colony formation as well as increased tumor cell death [61]. Potentially,

miRNAs or proteins involved in the ribosome scanning of 5’-UTR can alter _FUS1/TUSC2_ mRNA stability or its translation during progression from normal bronchial epithelium to malignancy

[54]. Cancer-specific epigenetic mechanisms involved in _FUS1_ gene suppression have also been reported. Generally, loss of a gene expression in tumors occurs ~10 times more frequently due

to CpG islands hypermethylation of the promoters than due to mutations [62]. Histone H3K9 methylation patterns (H3K9me1-3) are also responsible for gene silencing [63], whereas H3

acetylation leads to the opposite effect [64]. As for _FUS1_, no CpG methylation in the promoter was demonstrated in lung, nasopharyngeal, and breast cancers, even though promoter

hypermethylation in the neighboring gene _RASSF1_ was detected [65, 66, 47, 10]. However, partial promoter methylation of the _FUS1/TUSC2_ gene was reported in head-and-neck [67] as well as

in 20% of NSCLC cancers [68]. As for histone modifications, predominance of H3 acetylation (H3K9ac) over methylation (H3K9me3) was reported [66]. Physiological stimuli affecting _FUS1/TUSC2_

mRNA levels vary in their origin. The expression is reported to be downregulated by reactive oxygen species (ROS) [69, 70], and upregulated by hypoxia [58] and the factors stimulating

differentiation [71]. Summary of mechanisms regulating Fus1 levels is presented in Fig. 2. However, a fuller understanding of how _FUS1/TUSC2_ expression is regulated by physiological and

pathological stimuli is still lacking. PROTEIN MOTIFS AND POSTTRANSLATIONAL MODIFICATIONS OF FUS1/TUSC2 Human FUS1/TUSC2 is a small (110 aa) protein with an estimated MW of 12 kD. The

protein is basic and predicted to have a pI (isoelectric point) of 9.69 [10]. According to a computer-based modeling, FUS1/TUSC2 lacks transmembrane domains, is highly hydrophobic and

contains helix-coil domain secondary structures [53]. A 3D model for FUS1/TUSC2 protein is shown in Fig. 1B. Tertiary FUS1/TUSC2 structure is still debatable. Below are the functional motifs

identified in the protein sequence * 1. At the N-terminus, FUS1/TUSC2 contains a myristoylation signal (Met-Gly-X-X-X-Ser/Thr), and experiments confirmed that FUS1 is myristoylated [53]

(Fig. 1C). * 2. Motif-based profile scanning found a protein kinase A interaction site, an A-kinase anchoring protein interaction site, and a PDZ class II domain [53]. * 3. _In silico_

analysis revealed that protein fragment 54–65 aa is highly homologous to the EF-hand Ca2+-binding domains found in calmodulin as well as in mitochondrial proteins MICU1 and LETM1 [24, 25]

(Fig. 1C). * 4. The N-terminal fragment of FUS1/TUSC2 (positions 45–110) is 53% homologous to myristoyl-binding domain of recoverin, a typical Ca2+/myristoyl switch protein. Remakably, 9 of

11 key amino acids that form myristoyl-binding hydrophobic pocket of recoverin share similarity with the FUS1 protein [24, 25] (Fig. 1C). * 5. Fus1/Tusc2 of _C. elegans_ carries a bipartite

NLS (nuclear localization signal) (residues 84–101) and possesses weak similarity to DNA-directed RNA polymerase subunit A [10]. * 6. The N-terminus of Fus1/Tusc2 displays 40% similarity to

the DNA-binding domain of IRF7 [69]. * 7. The Fus1/Tusc2 with pos. 57–65 is identical to the 9 aa transactivation domains of transcription factors (TFs) p53, NFAT, and NFκB as predicted by

9aa TAD tool [72]. Myristoylation is extremely important for cell localization and functioning of the FUS1/TUSC2 protein [53]. N-myristoylation is a post-translational modification

consisting of the removal of the N-terminal methionine from a protein and ligation of the released NH2 group of glycine to the residue of myristic acid, a 14-carbon saturated fatty acid

(Fig. 1D) [73]. The resulting lipid tail participates in protein folding and anchoring myristoylated proteins to different membrane compartments, where they execute their functions (Fig. 1E)

[74, 73, 75]. A mutant form of FUS1/TUSC2 missing the myristoyl tail has a shorter half-life (6 h _vs_ 12 h) due to improper protein folding and increased proteasome degradation [53]. Also,

myristoylation-deficient FUS1/TUSC2 loses its characteristic mitochondria/ER localization and its abilities to induce apoptosis and suppress tumor cell proliferation in vitro. Importantly,

it also acquires the abilities to promote tumor growth and metastases in vivo [53]. Therefore, loss of myristoylation may be considered a key event that leads to insufficiency of Fus1

function, when compared to mutations and hypermethylation [14, 53]. The predicted Ca2+ EF hand binding motif and hydrophobic binding pocket of FUS1/TUSC2 prompted it to be classified as a

novel calcium/myristoyl switch protein (Fig. 1D) [24]. Its involvement in Ca2+-dependent signaling have been reported for different cell types including CD4+ T lymphocytes, kidney epithelial

cells, osteoclasts, and mouse embryonic fibroblasts [71, 23, 24, 23, 25]. The presence of myristoylation and Ca2+ binding motifs in one protein is pivotal for regulation of cellular

processes. In has been established that Ca2+/myristoyl switch proteins, in response to Ca2+ binding to EF-hand motifs, release their lipid myristoyl tail from a protein hydrophobic pocket

and anchor it to membranes during Ca2+ elevations (Fig. 1E) [74, 75]. The Fus1/Tusc2 protein can undergo other posttranslational modifications such as arginine mono-methylation (R9) [76],

phosphorylation (S50, site for PKA and RSK) [77], and acetylation (K93) [78]. Moreover, NetPhos 3.1 software (http://www.cbs.dtu.dk/services/NetPhos/) predicts phosphorylation at S4 (site

for CDC2), S6, T46, and T70 (PKC) (Fig. 1C). Thus, several functional protein motifs and a spectrum of potential posttranslational modifications facilitate Fus1/Tusc2 involvement in sensing

and execution of various cellular programs. FUS1/TUSC2 IN MITOCHONDRIAL CA2+ REGULATION MITOCHONDRIAL CA2+ TRANSPORT: MAIN PLAYERS MitoCa2+ transport is balanced by Ca2+ import and export.

The main route for mitoCa2+ entry is mitochondrial Ca2+ uniporter holocomplex (MCUcx), consisting of membrane channel protein MCU (_m_itochondrial _C_a2+ _u_niporter) and MCU regulatory

proteins MICU1-3, MCUb, MCUR1, EMRE [79,80,81]. MCU is an inner mitochondrial membrane protein that forms a pentameric channel with a selectivity filter for Ca2+ ions (a DIME motif) (Fig.

3A) [82, 83]. MCU becomes permeable for Ca2+ when ion concentration reaches 1-10 μM, which is only possible at the contact with the endoplasmic reticulum (ER) where Ca2+ is released _via_

inositol-1,4,5-triphosphate receptors (IP3Rs). The 1-10 μM Ca2+ threshold is set by the Ca2+-binding EF-hand motif of the mitochondrial protein MICU1 (_mi_tochondrial _C_a2+ _u_ptake_1_)

(Fig. 3A) [84, 85]. In the absence of MICU1, MCU transports Ca2+ at much lower cytoplasmic concentrations (hundreds of nM range) accompanied by mitoCa2+ overload [80]. However, other reports

suggested that the lack of MICU1 inactivates MCU in a Ca2+-dependent manner at a faster rate _via_ autoinhibition. Accordingly, MICU1 plays a gatekeeper role for MCU and prevents its

premature inactivation [86]. MICU1 heterodimerizes with its analogs, MICU2 and MICU3. Thus, MICU1/MICU2 heterodimer fine-tunes Ca2+ currents: MICU1 stimulates Ca2+ uptake at high cytosolic

Ca2+ level while MICU2 inhibits MCU at low cytosolic Ca2+ content [87]. The importance of MCU in tumor growth is based on its involvement in cell death. Malignant cells benefit from

reduction of mitoCa2+ uptake. This is especially important as tumor cells experience increased ROS production. Interestingly, MCU contains ROS sensing cysteines in its N-terminus. Their

oxidation promotes MCUcx assembly, persistent channel activity, and mitoCa2+ overload following by opening the permeability transition pore, which triggers cell death (Fig. 3B) [80].

However, for some breast cancers it was reported that activation of MCU promotes cancer cells motility, invasion, and growth [79]. So, it is not surprising that mitoCa2+ accumulation

undergoes regulation by tumor suppressors. Thus, p53 directly interacts with the Ca2+-ATPase in ER, promotes its negative oxidative modification, and stimulates enhanced Ca2+ release from ER

with further Ca2+ transfer to mitochondria _via_ the MCU protein (Fig. 3A) [88]. The lipid/protein phosphatase PTEN binds IP3R and, thus, counteracts PKB/Akt phosphorylating and inhibiting

IP3R; this results in a higher mitochondrial Ca2+ accumulation and apoptosis (Fig. 3A) [89]. On the other hand, down-regulation of the MCU mRNA by miR-25 is accompanied by increased cancer

cell survival and apoptosis resistance (Fig. 3A) [90]. Extrusion of Ca2+ from mitochondria into cytosol is mediated by NCLX (Na+/Ca2+/Li+ exchanger), a mitochondrial form of Na+/Ca2+

exchanger (mNCX), and Ca2+-binding EF-hand motif containing LETM1, a 2H+/Ca2+ exchanger (Fig. 3A) [79, 91]. Diminished NCLX activity leads to mitoCa2+ overload and elevated ROS production

due to the activation of Krebs cycle [92]. In a few tested non-tumor cell models, these events led to cell death. In cancer cells, however, although increased ROS blocked cell proliferation,

they also induced metastasis _via_ the ROS/HIF1α signaling axis [93]. Therefore, Ca2+ transients in mitochondria play a decisive role in the normal and tumor cell biology. POTENTIAL

MECHANISM OF MITOCHONDRIAL CA2+ REGULATION BY FUS1/TUSC2 PROTEIN Localization of Fus1/Tusc2 in mitochondria and the discovery of a Ca2+-binding domain in its structure [22, 24] suggested

that Fus1/Tusc2 could regulate mitoCa2+ accumulation. Indeed, Fus1 loss reduced mitoCa2+ accumulation resulting in retention of Ca2+ in cytosol [24, 23]. Like MICU1, Fus1 showed a dual

effect on mitoCa2+. Fus1-deficient cells had increased steady-state mitoCa2+ levels, while decreased cytoCa2+ levels. Moreover, during Ca2+ response, Fus1−/− mitochondria accumulated Ca2+ at

a faster initial rate than WT mitochondria and reached higher Ca2+ values at the peak of response. However, levels of mitoCa2+ declined faster during the recovery phase of Ca2+ response in

Fus1-deficient cells [24, 23, 25]. This effect of Fus1/Tusc2 deficiency on mitoCa2+ was partially alleviated by inhibition of mNCX responsible for mitoCa2+ extrusion [24]. Accordingly,

Fus1/Tusc2 may set a threshold for Ca2+ uptake similar to MICU1 and prevent mitoCa2+ accumulation at steady-state or rapid Ca2+ accumulation after initial rise in cytoCa2+ (Fig. 3C, left).

However, when cytoCa2+ concentration reaches high values, we propose that Fus1/Tusc2 binds Ca2+ ions, and releases its lipid tail that anchors protein to the mitochondrial membrane, thus

maintaining mtoCa2+ uptake. Finally, Fus1/Tusc2 inhibits mNCX and promotes maximal Ca2+ accumulation in mitochondria (Fig. 3C, right) [24, 23, 25]. When levels of cytoCa2+ start recovering,

drop in Ca2+ initiates reversed events: Ca2+ import _via_ MCU declines and mNCX accelerates Ca2+ export out of mitochondria. Although regulation of cytoCa2+ by mitochondria is complex,

usually deficiency in MCU translates into cytoCa2+ retention (Fig. 3D) [94]. Sustained cytoCa2+ results in prominent activation of Ca2+-dependent proteins, i.e., NADPH oxidase [95], CAMKII

[94] or Miro1 [80]. Initially, it was proposed that MICU1 regulates MCU-mediated Ca2+ currents _via_ Ca2+/myristoyl switch mechanism [96]. However, the MICU1 and MCU interaction is mediated

_via_ direct protein binding. At the MICU1 C-terminus, KQRLMRGL peptide represents an MCU-binding domain interacting with with DIME motif _via_ salt bridges [97]. Structural analysis of the

MCU N-terminal domain (NTD) surprisingly revealed unidentified lipid molecule bound to a hydrophobic protein surface formed by residues in the L1 loop, two helices (α2 and α3) and C-terminal

tail. This lipid was described as a linear lipid-like structure consisting of 13–16 carbon atoms that is similar to a tetraethylene glycol molecule [67]. It is noteworthy that myristic acid

(a substrate for N-terminal myristoylation) is a saturated linear long-chain lipid with a 14-carbon backbone complying with the features of an unidentified lipid. Therefore, Fus1/Tusc2

could regulate the activity of MCU _via_ a novel mechanism of inter-protein lipid tail exchange. We suggest that the myristic acid residue of FUS1/TUSC2 protein released from the hydrophobic

pocket after Ca2+ binding to EF-hands could interact with the hydrophobic surface of MCU and maintain its Ca2+ transporting function (Fig. 4A). This is likely in view of the fact that

Fus1/Tusc2 has the amino acid sequence KARGLWPF resembling MCU-binding domain of MICU1–3 proteins (Figs.1, 4B and C). Moreover, FUS1/TUSC2 can potentially interact with MCU _via_ its

inter-protein interaction site at the C-terminus (positions 81-96) (Figs. 3 and 4C). This mechanism needs further elucidation by functional and structural studies. PUTATIVE FUS1

PROTEIN-PROTEIN INTERACTION _VIA_ MYRISTOYL TAIL EXCHANGE An intriguing opportunity for protein-protein interactions was demonstrated by mutational analysis of FUS1/TUSC2 binding to a

pro-tumorigenic tyrosine kinase c-Abl [98, 99]. Initiated by discovery of a truncated form of FUS1/TUSC2 (deletion of C-terminal 83-110 aa) in tumor cells, Lin et al. showed that the

FUS1/TUSC2 81-96 aa C-terminal peptide linked with stearate inhibits c-Abl activity and facilitates its degradation. On the contrary, the FUS1/TUSC2 N-terminal peptide 1-80 aa, although

capable of binding to c-Abl, was unable to inhibit its kinase activity [98]. Like FUS1/TUSC2, c-Abl is a myristoylated protein. It is activated by releasing the myristoyl tail from the

hydrophobic pocket and conforming to an active open state, which allows c-Abl to bind its substrates _via_ the SH2-domain [100]. FUS1/TUSC2 mutational analysis showed that while the

N-portion of FUS1/TUSC2 binds to myristoyl-binding pocket of c-Abl, the C-terminal portion of FUS1/TUSC2 (containing c-Abl inhibitory peptide 81-96 aa) interacts with the ATP-binding site in

the N-lobe of c-Abl. Alternatively, FUS1/TUSC2 could interact with an activation loop in the C-lobe of c-Abl [99]. Initially, it was thought that myristoylation is exclusive for targeting

proteins to certain membrane compartments but, nowadays, this posttranslational modification is appreciated to play roles in protein folding and prevention from premature degradation [100,

101, 53]. A synthetic ligand for myristoyl binding pocket named GNF-5 maintains closed inactive conformation of c-Abl apo form or convert open conformation to a closed one [102]. Therefore,

one could speculate that the myristoyl tail of FUS1/TUSC2 inserted into the hydrophobic pocket of c-Abl inhibits its tyrosine kinase activity. This tail-pocket interaction can be an

important feedback loop in the network of interactions initiated by RTKs. Thus, platelet-derived growth factor receptor (PDGFR) stimulates signaling molecules such as PLCγ1, c-Src, and

c-Abl. In turn, PLCγ1 generates IP3 and promotes Ca2+ release from the ER [103]. We suggest that activation of FUS1/TUSC2 by Ca2+ leading to the release of its myristoyl tail could suppress

c-Abl activity. Noteworthy, c-Abl-deficient B cells exhibited reduced Ca2+ flux in response to antigen receptor or CD19 stimulation [104] reinforcing the concept of mutual regulatory loops

between FUS1/TUSC2 and c-Abl. CA2+ SIGNALING FINE-TUNED _VIA_ FUS1/TUSC2 IMPACTS CELL FATE Maintaining mitoCa2+ at moderate levels is important due to the effect of Ca2+ on ROS production

and cell death enhancing effect of Ca2+ overloading [53, 77]. The MICU1-3 proteins fine-tune MCU activity by setting a threshold to filter out mitoCa2+ elevations at low cytosolic Ca2+

levels (<350 nM) and cooperatively increasing MCU currents at high Ca2+ elevations (Fig. 3A) [89, 97]. Similarly, FUS1/TUSC2 may controls basal mitoCa2+ by inactivating MCUcx, which

prevents rapid initial Ca2+ accumulation and promotes adequate mitoCa2+ elevation (Fig. 3C) [23, 24, 23, 25]. This translates into appropriate activation of Krebs cycle and sufficient

formation of antioxidants (NADH, NADPH) maintaining ROS at low level. Accordingly, loss of antioxidant defense in _Fus1_-deficient cells [24, 25, 49, 69], would reduce cell proliferation,

tissue repair, and promote cell death. Indeed, MICU1 deficiency accompanies compromised liver regeneration after partial hepatectomy due to inflammation, overloaded mitoCa2+, blocked

proliferation, and increased necrosis of hepatocytes [105]. Compromised capability of _Fus1_−/− adult stem cells to repopulate tissues was reported in aged _Fus1_−/− animals (e.g., hair

follicles, thymus) [23] or younger mice after exposure to radiation (e.g., GI crypt epithelial cells, melanocyte stem cells) [49]. In vitro data suggest that Fus1 is involved in bone

remodeling shaped by bone deposition (osteoblasts) and resorption (osteoclasts) [106]. Silencing of _Fus1/Tusc2_ gene in bone marrow precursor cells in vitro diminished RANKL-induced

osteoclast differentiation without affecting osteoblast formation [71]. Thus, it is possible that _Fus1/Tusc2_ loss in vivo could also affect bone remodeling, shifting it towards bone

deposition that would reduce tissue repair potential [106] corroborating with accelerated aging and development of age-related disorders in _Fus1_−/− mice [23]. Thresholds in the mitogenic

signal transduction demand tight control of activation of proteins involved in cell proliferation. Malfunction of key tumor suppressors (e.g., E3 ligase PML) may switch cell fate from

senescence to malignant transformation [50, 107]. The ability of Fus1/Tusc2 to calibrate Ca2+ responses would translate into adequate cell responses based on the characteristics of input

signals such as signal strength. In this regard, Fus1/Tusc2 reminds other tumor suppressors (e.g. PTEN, Spry), which prevent cells from over-stimulation by mitogenic signals maintaining

their survival and responsiveness to proliferation signals [108,109,110,111,112]. REGULATION OF APOPTOSIS BY FUS1/TUSC2 Numerous studies demonstrated that _FUS1/TUSC2_ overexpression in

cancer cells that lack 3p21.3 or _FUS1/TUSC2_ gene/expression induces cell death [2, 46, 47, 53, 113, 114]. Co-expression of _FUS1/TUSC2_ and _p53_ synergistically increased apoptosis in

NSCLC cells. This synergistic effect was associated with the ability of Fus1/Tusc2 to down-regulate expression of MDM2, an E3 ubiquitin ligase that suppresses p53, and thereby stabilizes p53

levels. Importantly, combined effect of Fus1 and p53 co-expression required Apaf-1 that mediates mitochondria- and caspase3-dependent apoptosis(113). Additionally, in thyroid cancer cell

lines, overexpression of _FUS1/TUSC2_ increased levels of Smac/Diablo that blocks caspase regulatory inhibitors of apoptosis proteins and cytochrome _c_ [115]. Protein Chip array and

SELDI-TOF mass spectrometry revealed direct interaction between PDZ domains of FUS1/TUSC2 and Apaf [116] corroborating the link of FUS1 with the Apaf-1-mediated apoptosis. FUS1/TUSC2 IN

CELLULAR SENESCENCE CELLULAR SENESCENCE: MAIN PLAYERS Cellular senescence refers to cellular aging characterized by a highly stable cell cycle arrest accompanied by biochemical and

morphological alterations [117, 118]. The phenomenon of a limited number of cell divisions discovered by Leonard Hayflick in 1961 became known as the Hayflick limit [119]. A role of p53 in

senescence is evident from its effect on cell cycle arrest usually mediated by up-regulation of p21, an inhibitor of cyclin-dependent kinases CDK1, CDK2, and CDK4/6 required for the G1/S

transition [120, 121]. Senescence can be induced by oncogene overactivation, oxidative stress, genotoxic drugs, radiation, CDK suppression, demethylating and acetylating agents, etc. [121].

Cell cycle arrest accumulates unphosphorylated form of Rb protein, upregulating the lysosomal compartment and subsequently stimulating mTOR signaling (Fig. 5) [122]. mTOR signaling, in turn,

up-regulates mitochondrial biogenesis _via_ TF PGC1α. Increased mass and dysfunctional state of mitochondria enhance intracellular ROS and DNA damage. Downstream of this cascade, DNA damage

kinases (i.e., ATM) further activate the AKT/mTOR signaling axis stimulating PGC1α and creating a positive feedback loop stabilizing senescent state (Fig. 5) [117, 118]. DNA damage is

responsible for activation of the NFκB pathway regulating senescence-associated secretory phenotype (SASP); senescent cells secrete proinflammatory cytokines (IL-1β, IL-8, IL-6), chemokines

(MCP-1, CCL3, CXCL1), matrix metalloproteinases (MMP3, MMP9), etc. (Fig. 5) [123, 117, 118]. Moreover, SASP reinforces senescence _via_ autocrine pathway and induces it in neighboring cells.

Secreted cytokines and chemokines attract immune cells necessary for clearance of senescent cells (Fig. 5). However, accumulation of senescent cells results in the development of chronic

inflammatory disorders [124, 125, 121]. Initially considered an in vitro phenomenon, senescence was recently confirmed in vivo; transplantation of senescent cells from ear cartilage into

knee joint caused an osteoarthritis-like phenotype in mice [126]. Also, senescent fibroblasts can promote growth and proliferation of tumor cells _via_ secretion of SASP intermediates (e.g.,

fibroblast growth factors 10 and 19, IL-1β), epithelial-mesenchymal transition (IL6, MMP2-3), and immune evasion mechanisms (i.e., IL-6 drives accumulation of suppressive myeloid cells and

their activity) [124]. Therefore, senescence and SASP-mediated chronic inflammation could underlie the development of aging-related diseases including tumors. CA2+ SIGNALING AND SENESCENCE

Fine-tuned control of cellular Ca2+ signaling by tumor suppressors and oncogenes [127] established that increased cellular Ca2+ (cytosolic and/or mitochondrial) results in senescence or

apoptosis, whereas moderate levels of Ca2+ favor cell proliferation. Ca2+ regulates SASP via activation of calpain that converts pro-IL-1α into functional IL-1α(128). Another Ca2+ effector

is NFAT, a TF regulated by calmodulin/calcineurin complex that has both pro-proliferative and pro-senescent effects. In particular, NFAT induces p53 by suppressing expression of ATF3, a p53

negative regulator. NFAT acts as a pro-senescent factor by activating expression of IP3R2 that mobilizes Ca2+ from ER elevating cytosolic and mitoCa2+ levels [128]. IP3Rs are instrumental in

cancer cells, where the number of oncogenes and tumor suppressors cross their paths [127]. Pro-survival/pro-tumorigenic PKB/Akt phosphorylates and inactivates IP3R3. Tumor suppressor

protein PML binds to IP3R3 and recruits protein phosphatase PP2A to dephosphorylate IP3R3 leading to IP3R3-marked opening (Fig. 3A). Consequently, Ca2+ accumulation in mitochondria would

increase ROS production leading to a senescent state (Fig. 3B). It is noteworthy that abrogation of _MCU_ helps cells avoid senescence [128] and apoptosis, and promotes uncontrolled

proliferation, migration, and metastases [80]. FUS1/TUSC2-DEPENDENT PROCESSES MEDIATING CELLULAR SENESCENCE PROLIFERATION The role of Fus1/Tusc2 in the control of senescence appears to be a

part of its role as a TSG. _FUS1/TUSC2_ overexpression in tumor cells is associated with cell cycle arrest at G1/S [47] and G2/M [115] transition checkpoints. Deletion or silencing of

_Fus1/Tusc2_ accelerates proliferation of activated mouse CD4+ T cells [24] and human tumor cells [129]. Upregulation of miR-197 miRNA that increases _FUS1/TUSC2_ levels and, thus,

suppresses tumor metastasis was attributed to the ability of FUS1 to regulate proliferation of human glioblastoma cells [129]. GENOTOXIC STRESS In several models of cell injury, _Fus1_−/−

cells showed increased levels of genotoxic stress and senescence markers compared to wild-type counterparts. For example, after intraperitoneal injection of asbestos, infiltrating cells from

Fus1−/− mice had higher levels of γH2AX, DNA damage response molecule, and phosphorylated pro-inflammatory NFκB and ERK1/2. This was associated with increased number of macrophages and

accelerated accumulation of granulocytes in the peritoneal cavity. Among others, IL-1β, a signature cytokine of SASP, was up-regulated in peritoneal Fus1−/− cells. The increased sensitivity

to irradiation in Fus1−/− mice was accompanied by accelerated cell cycle arrest, aberrant mitosis, lack of proper DNA repair (mitotic catastrophe), early activation of p53, and death of

gastrointestinal crypt cells, which are especially susceptible to ionizing radiation [49]. Thereby, Fus1-deficient mice demonstrated enhanced cell damage upon challenging stimuli like other

TSG deficiency models (i.e., p27) [8]. CLEARANCE OF SENESCENT CELLS The most striking alteration in the chemokines/cytokines profile of _Fus1_-deficient mice was down-regulation of

RANTES/CCL5, a chemokine for T and NK cells [70]. Since T and NK cells are necessary for clearance of senescent cells [130], lack of CCL5 production in _Fus1__−__/−_ mice could lead to

accumulation of senescent cells during the course of lifetime and result in chronic inflammation, aging-related diseases, and malignant transformation. CONSEQUENCES OF FUS1/TUSC2 DEFICIENCY

AT ORGANISMAL LEVEL FUS1/TUSC2-ASSOCIATED SYSTEMIC PATHOLOGIES Several Fus1-dependent pathologies developing in mice have been reported following the targeted _Fus1_ inactivation. These

could be divided into two groups: SPONTANEOUS PATHOLOGIES DEVELOPED IN YOUNG OR MIDDLE-AGED MICE AND PROGRESSING WITH TIME * a. Chronic systemic inflammation [23]. * b. Progressive

development of SLE-like autoimmune disease in some mice (incomplete penetrance) (vasculitis, glomerulonephritis, anemia, circulating autoantibodies) [22]. * c. Increased frequency of

spontaneous vascular tumors [22]. * d. Preponderance of aging signs that include lordokyphosis, absence of vigor, diminished hair regrowth, reduced sperm count and motility, enlarged seminal

vesicles, and compromised stem cells self-renewal [22, 23]. * e. Early development of aging-associated diseases: * 1. Premature progressive hearing loss (higher threshold for sound

intensity, longer latency to respond to sound, and smaller amplitude in auditory brainstem responses (ABR) waves, compared to wild-type mice [50]. The hearing loss corresponded with impaired

PTEN/Akt/mTOR pathways in cochlear cells and was ameliorated by administration of N-acetyl cysteine, an antioxidant agent [50]. * 2. Impairments in olfactory and spatial memory at a

relatively young age (4-5 months old), as indicated by habituation test, hidden cookie test, and Morris water-maze test [51]. INJURY-INDUCED PATHOLOGIES IN YOUNG MICE * a. Higher sensitivity

to γ-irradiation [48, 49]. * b. Higher sensitivity to peritoneal asbestos injury [70]. * c. Resistance to _A. baumanii_ lung infection [21]. Most likely, defects in common Fus1-dependent

mechanism(s)/pathways that are discussed below underlie these pathologies. THE MTOR PATHWAY ACTIVATION _Fus1_−/− mice showed prominent mTOR signaling activation and oxidative stress,

signature hallmarks of senescence and early aging. In young _Fus1_−/− mice, cochlear cells are distinguished with reduction of antioxidant enzymes (mitochondrial SOD2, PRDX1) and concomitant

activation of the Akt/mTOR pathway (decrease in PTEN levels, up-regulation of phospho-Akt and S6). This molecular pattern was associated with low-grade chronic inflammation observed in bone

marrow cells in the temporal bone surrounding the cochlea [50]. During senescence, activation of Akt/mTOR (for example, _via_ DNA damage/ATM pathway) stimulates PGC1α followed by

up-regulation in mitochondrial biogenesis. Mitochondria-derived ROS damage DNA and, thereby, maintain mTOR activation. Additionally, mTOR regulates translation of mRNA related to SASP _via_

MAP kinase-activated protein kinase 2 phosphorylating protein ZFP36L1 responsible for mRNA degradation (117). Therefore, it is not surprising that chronic inflammation accompanies senescent

phenotype of cochlear _Fus1_−/− cells [50]. OXIDATIVE STRESS Increased ROS stabilize p21, a p53 activator and key suppressor of cell proliferation in senescence, and inhibit autophagy 23.

Increased ROS production was evident in _FUS1/TUSC2_-deficient head-and-neck cancer cells JHU012 [70] and in mouse splenocytes [24]. Additionally, cochlear cells from _Fus1_−/− mice

demonstrated up-regulation of antioxidant defense proteins (Prdx1) in steady-state epithelial cells as well as gradual decrease in the expression of Sod2 and Prdx1 [50]. Further, _Fus1_−/−

primary mouse embryonic fibroblasts and immortalized kidney epithelial cells showed defects in respiration such as significantly decreased maximal mitochondrial respiration and respiratory

reserve capacity, likely due to down-regulation of mitochondrial respiratory proteins [127]. However, upon challenging conditions (i.e., irradiation) _Fus1_-deficient cells displayed delayed

up-regulation of Sod2, a mitochondrial form of antioxidant superoxide dismutase [49]. Treatment with antioxidants (Tempol, pyroxidamine) restored Sod2 expression and significantly improved

survival of whole-body irradiated Fus1-deficient mice [48]. Another antioxidant, N-acetylcysteine, rescued expression of Prdx1 and respiratory chain compounds, restored normal mitochondrial

morphology, and prevented progression of hearing loss in _Fus1_−/− animals [50]. DISRUPTION OF MITOCHONDRIAL CA2+ HOMEOSTASIS Increased ROS production in _Fus1/Tusc2_-deficient tissues may

derive from Fus1 ability to regulate mitoCa2+ transport [23,24,25]. Indeed, inhibition of MICU1 _via_ Akt/PKB phosphorylation increased ROS production and downstream Akt/PKB activation [85,

90]. The deficiency in mitoCa2+ leads to elevated cytosolic Ca2+ and activation of Ca2+-dependent proteins in the cytosol. For example, Ca2+-dependent stimulation of Miro1 results in the

remodeling of long filamentous mitochondria into globe-shaped mitochondria, step prerequisite for autophagosomal degradation [80]. Indeed, presence of globule-shaped giant mitochondria in

_Fus1_-deficient epithelial and cochlear cells point to preferred mitochondrial fission in these cells [24, 51]. This process should activate mitophagy, but in _Fus1_−/− mice it rather leads

to accumulation of nonfunctional mitochondria. _Fus1_−/− cochlear cells displayed a major down-regulation in the expression of PTEN-induced kinase-1 (PINK1) [51], a sensor of mitochondrial

quality control, which directs dysfunctional mitochondria towards autophagy/mitophagy [131]. Importantly, PINK1 protects cells from oxidative stress and premature senescence [132].

Therefore, _Fus1_-deficient cells accumulate dysfunctional mitochondria leading to further increase in ROS production and senescence [50]. INFLAMMATION Transcriptomic analysis of _Fus1_−/−

CD4+ T cells revealed that at the basal level T lymphocytes significantly up-regulate gene expression of secretory pro-inflammatory markers such as _Mmp8-9_, _S100a8-9_, _Lcn2_, _Ltf_,

_Retnlg_ [24]. Some of these markers (S100a8-9, MMP8-9) are signatures for SASP [133,134,135] and associate with chronic inflammation observed in _Fus1_-deficient mice [23, 50, 70].

Persistent oxidative stress leads to chronic inflammation, whereas senescence limits stem cell turnover culminating in early aging. Therefore, _Fus1/Tusc2_ loss links systemic aging to early

senescence, geriatric diseases as well as to tumor escape from immunosurveillance [136]. CONCLUSIONS AND CLINICAL RELEVANCE The Fus1-mediated cellular homeostasis is at the crux of its

tumor suppressor, anti-inflammatory, and anti-aging activities. Developing _FUS1_-based genetic therapies for cancer patients became an apparent step after revealing its TSG properties. In

preclinical studies, intra-tumoral injection of cationic liposome nanoparticles complexed with plasmid DNA encoding _FUS1/TUSC2_ gene (REQORSA or quaratasugene ozeplasmid formely known as

Oncoprex) significantly inhibited growth of human NSCLC cells H1299 and A549 in subcutaneously inoculated mice. Administered intravenously, REQORSA suppressed metastases and extended

survival of tumor-bearing mice [114]. Phase-I clinical trial reported the effective dosage and safety of REQORSA[13]. Conventional therapy for metastatic NSCLC currently uses epidermal

growth factor receptor (EGFR) tyrosine kinase inhibitors such as erlotinib, gefitinib, and osimertinib [137]. Beside toxicity, this type of therapy often leads to treatment-resistant cancer

as a result of selecting tumor cell clones carrying mutations. Thus, continuous treatment with erlotinib prescribed to NSCLC patients with overexpression or mutations in EGFR (deletion in

19th exon or L858R point mutation), leads to accumulation of cell clones with T790M mutation in EGFR or up-regulation in other growth receptor signaling pathways (e.g., HGFR, RBB3/PI3K)

leading to drug resistance. Osimertinib allows to overcome this issue as it can block EGFR mutated at T790M site; however, resistance to this treatment can also develop after losing tumor

cells with T790M [137]. Therefore, combination of EGFR inhibitors with other treatments affecting upstream signaling pathways involved into drug resistance is highly desirable. Ability of

Fus1/Tusc2 to suppress receptor and non-receptor kinases as well as sensitize NSCLS cells to chemotherapeutics [14, 98, 138, 139] makes this tumor suppressor a promising therapeutic

candidate for combinatorial therapy. Currently, two clinical trials based on combination of Fus1-gene based drug REQORSA and EGFR inhibitors erlotinib

(https://clinicaltrials.gov/ct2/show/NCT01455389) and osimertinib (Acclaim-1, https://clinicaltrials.gov/ct2/show/NCT04486833) are underway for treatment of NSCLC patients. Ongoing

revolution in immunotherapy started with a clinical introduction of checkpoint inhibitors significantly improved therapeutic outcomes in patients with different types of cancer (melanoma,

breast cancer, colon cancer, Hodgkin lymphoma, etc.) [15,16,17]. However, effectiveness of immunotherapeutics is limited due to evasion of tumor cells from immunosurveillance mechanisms

(e.g., up-regulation of TIM-3 immune suppressive molecules on cancer cells after PD-1 blocking therapy) eventually leading to tumor resistance similar to chemotherapy [18, 19]. Thus,

treatments with drugs targeting pathways upstream of drug resistance would be beneficial. Overexpression of _FUS1/TUSC2_ down-regulates mTOR signaling, which stimulates PD-L1, an

immunosuppressive ligand up-regulated in many types of tumors including NSCLC. _FUS1/TUSC2_-induced decrease of PD-L1 expression in response to its main inducer, IFN gamma, modifies tumor

microenvironment, unleashes T and NK cells from inhibition, and allows effective use of PD-1 blockers [24, 140]. REQORSA and anti-PD-1 combined therapy demonstrated significantly stronger

immune response than individual therapies. This resulted in the development of favored anti-tumor response (Th1 differentiation, NK and CD8 + CTL recruitment) and down-regulation of immune

suppression signatures (PD-1, CTLA-4, TIM-3) [20]. Recently, a clinical trial Acclaim-2 using REQORSA (also called GPX-001) combined with PD-1 blocking Abs (Pembrolizumab) in treated

non-small lung cancer patients has been launched [https://clinicaltrials.gov/ct2/show/NCT05062980]. Thus, studies initiated about 40 years ago to dissect chromosomal aberrations in lung

cancer cells culminated in a new promising gene therapy aimed to battle most aggressive lung cancer stages. In addition, Fus1/Tusc2-mediated anti-inflammatory and anti-aging activities

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Scholar  Download references ACKNOWLEDGEMENTS This work was supported by funds from the following National Institutes of Health (NIH) grants: U54 CA163069 (AnS), U54 MD007593 (AnS, AkS,

SSC), SC1 CA182843 (AnS), and SC1 CA182843-07S1 (AnS). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in

or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. There was no role of the funding bodies in the design or writing of the

manuscript. No writing assistance was utilized in the production of this manuscript. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Biochemistry, Cancer Biology, Neuroscience

and Pharmacology, School of Medicine, Meharry Medical College, Nashville, TN, USA Roman Uzhachenko, Akiko Shimamoto, Sanika S. Chirwa & Anil Shanker * School of Graduate Studies and

Research, Meharry Medical College, Nashville, TN, USA Alla V. Ivanova * Host-Tumor Interactions Research Program, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN, USA

Anil Shanker * Vanderbilt Institute for Infection, Immunology and Inflammation, Vanderbilt University, Nashville, TN, USA Anil Shanker * Vanderbilt Memory and Alzheimer’s Center, Vanderbilt

University, Nashville, TN, USA Akiko Shimamoto & Anil Shanker * Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA Sergey V. Ivanov Authors * Roman

Uzhachenko View author publications You can also search for this author inPubMed Google Scholar * Akiko Shimamoto View author publications You can also search for this author inPubMed Google

Scholar * Sanika S. Chirwa View author publications You can also search for this author inPubMed Google Scholar * Sergey V. Ivanov View author publications You can also search for this

author inPubMed Google Scholar * Alla V. Ivanova View author publications You can also search for this author inPubMed Google Scholar * Anil Shanker View author publications You can also

search for this author inPubMed Google Scholar CONTRIBUTIONS RU conceived and prepared the first draft. AVI, SSC, AkS, SVI, and AnS reviewed and edited the manuscript. RU designed figures.

All authors read and approved the final manuscript for publication. CORRESPONDING AUTHORS Correspondence to Alla V. Ivanova or Anil Shanker. ETHICS DECLARATIONS CONFLICT OF INTEREST The

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ARTICLE Uzhachenko, R., Shimamoto, A., Chirwa, S.S. _et al._ Mitochondrial Fus1/Tusc2 and cellular Ca2+ homeostasis: tumor suppressor, anti-inflammatory and anti-aging implications. _Cancer

Gene Ther_ 29, 1307–1320 (2022). https://doi.org/10.1038/s41417-022-00434-9 Download citation * Received: 13 October 2021 * Revised: 22 December 2021 * Accepted: 28 January 2022 *

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