It has been over forty years since the isolation of the first human oncogene (HRAS), a crucial milestone in cancer research made possible through the combined efforts of a few selected research groups at the beginning of the 1980s. Those initial discoveries led to a quantitative leap in our understanding of cancer biology and set up the onset of the field of molecular oncology. The following four decades of RAS research have produced a huge pool of new knowledge about the RAS family of small GTPases, including how they regulate signaling pathways controlling many cellular physiological processes, or how oncogenic mutations trigger pathological conditions, including developmental syndromes or many cancer types. However, despite the extensive body of available basic knowledge, specific effective treatments for RAS-driven cancers are still lacking. Hopefully, recent advances involving the discovery of novel pockets on the RAS surface as well as highly specific small-molecule inhibitors able to block its interaction with effectors and/or activators may lead to the development of new, effective treatments for cancer. This review intends to provide a quick, summarized historical overview of the main milestones in RAS research spanning from the initial discovery of the viral RAS oncogenes in rodent tumors to the latest attempts at targeting RAS oncogenes in various human cancers. Almost twenty years before the identification and isolation of the first human RAS oncogene, JJ Harvey isolated a transforming retrovirus from leukemic rats that was capable of producing sarcomas in infected rodents. The virus was named Harvey-MSV (Murine Sarcoma Virus) and shown to be the result of the recombination between retroviral genomes and a cellular rat gene  that was responsible for the transforming ability of this virus , and subleotides . From 19Alu) sequences to identify and clone the first human oncogene , which happened to be a human homolog of the previously known viral Harvey-RAS oncogene [The greatest step forward in our understanding of the origin of human cancer and the role of the RAS GTPases in tumor development came at the beginning of the 1980s, with a flurry of independent reports produced by several different labs of the US east coast  . Workingoncogene ,8,9. Soooncogene . Finallyoncogene ,11. In poncogene ,13,14.The most striking discovery concerning the human RAS oncogenes and their relevance for the mechanisms of carcinogenesis was the surprising observation (at that time) that all three human RAS oncogenes became oncogenically activated through the acquisition of a single point mutation affecting one of two main hot spots, located around codons 12 or 61 of their primary nucleotide sequences ,16,17.Following the seminal observations establishing single point mutations as the oncogenic switch for RAS genes, extensive screenings from many laboratories worldwide analyzing different tumor types and stages confirmed the specific presence of mutated RAS oncogenes in a large variety of specific hematological and solid tumors  (early review in ). In thiFrom a mechanistic point of view, some reports were also relevant as they showed that simple RAS mutations are not enough for malignant transformation of primary fibroblasts. In particular, transfection studies using RAS oncogenes showed that fibroblast immortality is a prerequisite for transformation by the HRAS oncogene , and thaThe discovery and isolation of RAS genes was immediately followed by the characterization of their transcriptional and translational products. In this regard, this was relevant as it led to, for example, the discovery of alternative splicing of the K-RAS transcripts in mice , a discoThe development of expression vectors and experimental procedures allowing the exogenous expression and the purification of large amounts of activated RAS proteins contributed greatly to their subsequent biochemical characterization. These early biochemical studies using purified RAS proteins produced the crucial discovery that RAS is a GTPase and that the oncogenic mutations produce a strong reduction of their intrinsic GTPase activity , revealiAnother important milestone in RAS research was the discovery of post-translational modifications at the C-terminal region of RAS proteins which targeted these otherwise hydrophilic proteins to the plasma membrane ,27. WherAfter the initial isolation and characterization of the human RAS oncogenes in the first half of the 1980s, the search for related genes and gene products using specific antibodies and nucleic acid probes gave rise, during later years, to the identification and characterization of a wide superfamily of related, monomeric small GTPases sharing structural and phylogenetic similarities with the original, H, N and KRAS oncogenes and acting as signaling molecular switches controlling the activation of many essential cellular functions. The three canonical HRAS, NRAS and KRAS oncogenes represent a very small part of the RAS subfamily, whose members act in control of processes of proliferation and differentiation. The members of the more distantly related RHO/RAC, ARF, or RAB subfamilies are known to participate in the regulation of processes of intracellular vesicle movement and cytoskeletal organization (reviewed in ). FigureMacaca mulatta,Cebus capucinus)), rodents ), as well as mink and horses [Saccharomyces cerevisiae, Schyzosaccharomyces pombe) [Drosophila melanogaster, Caenorhabditis elegans) [Xenopus laevis [Analysis of the expression of RAS and RAS-related genes along the evolutionary scale soon demonstrated that the RAS GTPases are widely expressed in all eukaryotic cells and organisms. Thus, early screenings using antibodies generated against viral RAS proteins immediately showed the expression of related proteins in many different mammalian species including primates  ,35, inveelegans) ,37 or los laevis . Interess laevis ).In the last quarter of the past century, multiple separate contributions from different labs around the word provided a multifaceted, multipronged path of research and discovery that eventually produced seminal experimental advances and conceptual milestones regarding the basics of RAS structure, function and biology . The folXenopus oocytes and its associated phosphorylation events [The discovery and characterization of the human RAS oncogenes and the demonstration of their involvement in tumorigenesis set up the starting point for a race to uncover their role in different types of cancer and to find the signaling events that regulate, and are regulated by, these small GTPases. Between 1984 and 1987, most published reports on RAS function focused on analyzing its causal role in different types of human cancer, but very few described mechanistic aspects of RAS regulation of cellular signaling. Highly relevant in this regard were the initial discoveries that uncovered the participation of RAS proteins in signal transmission from growth factor receptors showing that EGF stimulates guanine nucleotide binding to RAS proteins  but thatn events ,42, or cn events ,44.A major advance in RAS-signaling biology derived from the discovery in 1987 of a novel yeast gene whose mutations triggered similar effects to RAS mutations in Saccharomyces cerevisiae. The gene was named CDC25 and was proposed to be a cellular activator of RAS GTPases by regulating nucleotide binding to RAS proteins . A few yThe most recent studies have demonstrated that the SOS GEFs are the most widely expressed and functionally relevant RAS activators in Metazoa, having a critical relevant role not only in individual cells but also in the control of organismal viability and homeostasis (rev. in ). In conXenopus oocytes, as well as from mouse and human cells, a protein was identified that was capable of multiplying by many times the intrinsic GTP hydrolytic activity of cellular RAS proteins. It was also observed that the oncogenic mutations rendered RAS proteins insensitive to the effects of this factor, which was named “GTPase Activating Protein” (GAP) [A few months after the discovery of CDC25, another important finding further clarified how the cycle of RAS activation/deactivation is regulated. Using extracts from n” (GAP) . Shortlyn” (GAP) . Parallen” (GAP) . Fourteen” (GAP) , the GAPn” (GAP) ).By the end of the 1980s it was already clearly established that RAS proteins are located at the plasma membrane, where they receive and transmit signals from surface growth factor receptors, but the intermediate link between the surface receptors and RAS activation was still missing. The first insight in this regard came with the discovery of GRB2, an adaptor protein that was shown to bind to phosphorylated tyrosine residues of the intracellular domain of transmembrane receptors that had been activated by agonist binding . The folEarly comprehensive mutagenesis studies carried out in different laboratories allowed an initial identification of “essential” and “dispensable” functional domains located along the primary amino acid sequence of RAS proteins. For example, since mutations within a region stretching from position 32 to 40 greatly reduced the biological activity of HRAS proteins, this region was postulated as an essential domain responsible for the interaction of RAS with its effectors . Later oThe first crystallographic structure of a RAS protein  was available in 1988 and identified four α-helixes and six β-sheets connected by nine loops . AnalysiBy 1993, the backbone of the upstream signaling processes leading to RAS activation had already been described. However, the signaling events elicited after RAS activation were not yet completely understood. For example, we mentioned above that the first protein defined as a RAS effector was p120RASGAP , but it After the discovery of RAS effectors, the key question to be answered was how downstream signaling managed to translate the activation of each effector into specific elicited cellular responses. Regarding RAF effectors, prior reports showing MEK activation by c-RAF  and actiThe following subsections describe several additional advances on RAS biology that have been produced in more recent years, thus leading to an improved understanding of RAS regulation and function in the plasma membrane and other intracellular domains.In addition to the early identification of palmitoyl and farnesyl binding to the C-terminal region, an increasing number of later reports have also shown that modulation of RAS activity is far more complicated and may rely also on many other covalent modifications. For example, whereas phosphorylation of RAS by PKC was discovered long ago , more reConsistent with functional differences among the isoforms, it was also shown that HRAS or NRAS, but not KRAS, can be mono- or di-ubiquitinated, and this modification targets these two isoforms to endosomes . FurtherRAS glutathiolation in cysteine 118 has also been described as a posttranslational modification affecting RAS function that may provide a functional link between RAS proteins and cellular oxidative stress . RAS provia the RAS-ERK pathway [Besides the intramolecular, covalent modifications of the RAS proteins, other regulatory mechanisms involving inter-molecular interactions have also been discovered in the last two decades. Early in RAS history, it was reported that RAS proteins are able to form homo-oligomers in vivo , but thi pathway . Importa pathway , and tha pathway ,117.Another very relevant finding regarding the regulatory mechanisms of RAS activation was the discovery of an allosteric binding site for RAS in SOS1, showing that the specific binding of an activated RAS.GTP molecule to SOS1, in a region independent of the catalytic region responsible for nucleotide exchange, results in a significant increase in the overall exchange activity on other RAS molecules through the establishment of a positive feedback loop ensuring that many more inactive RAS molecules are then processively activated by the same allosterically activated SOS molecule . MutatioThe capacity to signal from endomembranes is another relevant concept added to our understanding of RAS biology and function in the last 20 years. Although the location of RAS proteins in the endoplasmic reticle (ER) and Golgi was known very early, it was thought to be merely an intermediate step during protein maturation and transport to the plasma membrane. This notion was revoked in 2002 by experiments using RAF1 RBD probes linked to GFP to show that a portion of RAS found in ER and Golgi is bound to GTP and able to interact with downstream effectors . TherefoC. elegans [Another relevant conceptual advance regarding RAS regulation and function was the discovery of Let7, a microRNA capable of controlling RAS expression in  elegans . Let7 is elegans ).The crucial role of RAS genes and proteins regarding the control of normal eukaryotic cell proliferation was demonstrated early on by means of experiments showing that the microinjection of neutralizing RAS antibodies inhibited the initiation of S-phase in NIH 3T3 fibroblasts cells, indicating that RAS was essential for G1-S progression in the cell cycle . Besides controlling cellular proliferation and progression of the cell cycle, RAS proteins have also been found to regulate many other normal relevant cellular processes, including differentiation. For instance, viral RAS oncogenes were shown to alter differentiation of cultured epidermal cells , and humMany other physiological cellular processes are also regulated by RAS proteins. For example, RAS proteins have been shown to induce expression of the autocrine motility factor, a cytokine responsible for cell migration  and to cgain-of-function (hyperactivating) mutations that may occur in canonical RAS family members or other components of RAS signaling pathways. In fact, RAS signaling alterations constitute a common cause of cancer (somatic oncogenic mutations) but may also trigger a wide variety of other human illnesses, either as causal mutations or as symptom-related changes, such as the developmental defects caused by inherited gain-of-function mutations of various members of RAS signaling pathways.The critical relevance of the RAS GTPases in the control of normal cellular functions is further underscored by the many different deleterious effects that may appear in individual cells and living organisms as a consequence of the alterations of normal RAS signaling and functions caused by the The demonstration of the activation of the MAPK cascade by RAS, together with previous findings showing that oncogenic RAS mutants trigger increased MAPK phosphorylation and activity independently of receptor activation , indicatloss-of-function in many human malignancies. In fact, the role of PTEN as a tumor suppressor was known before its role as an inhibitor of the PI3K pathway [Alterations in other branches of RAS downstream signaling pathways have also been linked to cancer. In particular, aberrant PI3K signaling is frequently seen in human malignancies as shown by the detection of oncogenic PIK3CA mutations in colon, brain and other cancer types, including breast and endometrial tumors . Overexp pathway ,140. Another important concept, gleaned from studies on RAS signaling in cancer, is the notion of mutual exclusivity among the potential oncogenic mutations that may occur in different components of a shared linear signaling pathway. Indeed, the experimental observation has shown that the co-occurrence of oncogenic mutations in components of the RAS/RAF/MEK/ERK signaling pathway is practically non-existent, probably due to the fact that such co-expression leads to oncogene-induced senescence. This was first observed in human melanoma, where BRAF and NRAS mutations are more frequent but never concur in the same cancer , and is All RAS downstream effector signaling pathways have been associated with tumorigenesis. However, because of their crucial roles in regulation of proliferation, growth, cell migration, metabolism and apoptosis, the RAS-RAF/MEK/ERK and the RAS-PI3K/AKT signaling axes are believed to be most relevant for cancer development and, therefore, most therapeutic efforts so far have been focused on these two particular pathways .gain-of-function germline mutations in RAS-GEFs, such as SOS1, are responsible for another related defect: designated Noonan syndrome. In this case, the increased GEF activity of a mutated SOS1 would be responsible for the hyperactivation of the signals coming from the RAS/ERK pathway [The bulk of the experimental data accumulated during the last decades of the 20th century established the causal role of somatic RAS mutations in the development of a variety of sporadic human tumors appearing during adult life. Conversely, more recent research from the first decade of the 21st century produced the somewhat surprising discovery of the occurrence of inherited, germline RAS mutations in association with a number of distinct hereditary familial developmental syndromes now collectively known as RASopathies or cardio-facio-cutaneous-syndromes . Mutatio pathway . Various pathway ).Finally, other recent reports have also found evidence supporting the implication of altered RAS signaling in non-tumoral illnesses including diabetes, Alzheimer’s, intellectual disability or schizophrenia, among others ,151,152.G12V mutation were shown to harbor teratocarcinomas developed early during embryonic development, thus pointing to the lethality of germline RAS mutations [Genetically modified murine strains are highly instrumental in analyzing the physiological and pathological roles played by specific genes/proteins in different biological contexts. This was quickly made evident for RAS oncogenes when the first transgenic mice bearing an HRASutations . Afterwautations  and for utations . G12V and KRASG12D transgenes provided initial, direct evidence of the role of RAS oncogenes in cancer initiation and development by demonstrating the critical requirement of RAS oncogenes for tumor maintenance and the regression of tumors after RAS transgene expression was silenced, even in the absence of tumor suppressors such as INK4a or p53 [G12D transgene to develop mouse lung tumors resembling human NSCLC and also pancreatic tumors [Early seminal reports analyzing mouse strains harboring HRASa or p53 ,157. Sepc tumors . Other rc tumors .−/−/NRAS−/− knockout mice were also viable and presented no obvious phenotypic alterations [Whereas the study of transgenic mice produced significant, initial insights regarding RAS tumor biology, the generation and analysis of knockout mouse models has been crucial for understanding the normal physiological role of RAS proteins. The first report of a RAS knockout described that ablation of NRAS expression by gene targeting did not produce any gross phenotype, indicating the dispensability of NRAS for mouse development, fertility and growth . In conterations .+/−/NRAS−/− die either during embryonic development or soon after birth, showing that the viability of adult NRAS KO mice is dependent upon a fully normal dose of KRAS expression [−/−/NRAS−/− double KO mice were always obtained in crosses between HRAS KO and NRAS KO mice, and the resulting adult DKO showed low fertility rates, suggesting overlapping functionalities between HRAS or NRAS regarding embryonic development, postnatal growth and fertility [−/−/NRAS−/− DKO pups die between P0 and P2 due to defects in lung maturation, demonstrating functional redundancy for HRAS and NRAS in the last stages of lung development, where KRAS could not replace their function [Despite the strong evidence for the functional specificity of the H, N and K RAS genes, detailed analysis of the biology of different RAS KO strains point also to at least some partial functional overlapping among these RAS isoforms in vivo. For example, KRASpression . Similarertility . Consistfunction . It was function . −/−/KRAS−/− DKO embryos die in utero at earlier times than single KRAS KO embryos, suggesting overlapping functionalities of HRAS and KRAS, at least between E9.5 and E11.5. Consistently, the same report showed that expression of a human HRAS transgene under its natural promoter was able to rescue the lethality of KRAS KO mutant embryos, even in the context of a triple HRAS/NRAS/KRAS knockout [Other reports support the existence of overlapping functions between HRAS and KRAS. In particular, it was shown that double HRASknockout . These oknockout . Altogetknockout .The early transgenic mouse models carrying RAS mutations clearly demonstrated the causal relation between RAS oncogenes and tumor development but were not optimally designed to analyze the fine mechanistic and biological details related to in vivo tumor development of human cancers under different biological contexts. To overcome these limitations, new targeting strategies, allowing spatiotemporal induction of oncogenic mutations expressed under their native RAS gene promoters or breeding with genetically modified mice carrying other relevant mutations, have been later developed to allow the generation of rodent cancer models better recapitulating the behavior of human cancers.V12G transgenic mouse model that was crossed with a KO mouse strain lacking the tumor suppressor INK4a to generate offspring that developed malignant melanoma, but the skin lesions disappeared when doxycycline treatment was stopped, showing a critical role of RAS for tumor maintenance [A seminal report in this regard used a doxycycline-inducible HRASntenance .G12D in type II pneumocytes under the control of doxycycline developed lung tumors upon doxycycline treatment (indicating that KRAS mutations are necessary for tumor initiation), but the tumors regressed upon doxycycline removal . Furthermore, the tumors were more aggressive when the KRAS4bG12D mice were crossed with strains lacking tumor suppressors such as p53 or INK4A/ARF, but even maintenance of these tumors was dependent on a sustained KRAS4bG12D expression [This notion was later confirmed and extended in a model of lung cancer where transgenic mice expressing murine KRAS4bpression .G12D only in the lungs. In addition, these models allowed also the analysis of the stages of lung tumor progression . Consisn of p53 .Cre;LSL-KRASG12D and P48+/Cre;LSL-KRASG12D mice developed pancreatic intraepithelial neoplasias (PanIN) which evolved to PDAC and metastatic disease, a tumor evolution resembling human PDAC, but the tumor incidence in these mice was very low [+/LSLG12Vgeo;Elas-tTA;TetO-Cre), the oncogenic KRAS mutation was induced upon doxycycline treatment. These mice developed PDAC with a very similar pattern to the Cre;LSL-KRASG12D and P48+/Cre;LSL-KRASG12D mice, and the tumors were more aggressive in the absence of the p53 or INK4a/ARF tumor suppressors. Interestingly, pancreatic acinar cells of adult mice were resistant to oncogenic transformation by the KRASG12D oncogene, even in the absence of p53 [RAS mutation frequency is highest in pancreatic ductal adenocarcinomas (PDAC). Using a KRAS transgene expressed under control of pancreas-specific promoters (PDX-1 or P48), both PDX-1very low . In a lae of p53 ,171.−/− KO mouse strain, showing that genetic lesions found in human melanoma are able to induce malignant melanoma in mice [A mouse model of melanoma was generated by introducing an NRAS transgenic allele under the control of the tyrosinase promoter in a INK4a in mice .L1 mice with another mouse strain carrying the Cre recombinase under control of the Villin promoter. These mice developed colon cancer only in the proximal colon, showing that the molecular events leading to CRC are different depending on the region of the colon [G12D floxed allele and with a tamoxifen-inducible Cre recombinase model. The resulting mice developed CRC in a tamoxifen dose-dependent manner, with tumors being indistinguishable from human CRC [A murine model of KRAS induced colorectal carcinoma (CRC) was generated crossing the KRAShe colon . A more uman CRC .Mouse models of cancer have been highly instrumental to better understand tumor progression in humans and are also useful tools for preclinical studies analyzing the therapeutic usefulness of drugs previously developed and tested in vitro or on cell cultures. The development of complex strategies producing mouse models growing tumors that closely resemble human cancer has allowed the generation of several mouse strains usable as preclinical models in drug development and have given new insights to the role of RAS GTPases in human malignancies .There are also specific animal models allowing the study of mutations in the RAS pathway that are involved in RASopathies. The first such model involved genetic targeting of the NF1 locus to generate mice that developed symptoms very similar to human neurofibromatosis . Corresphttps://www.clinicaltrials.gov accessed on 5 April 2021, for more details). The initial search for therapeutic approaches targeting oncogenic RAS in cancer was for the most part a sad history of trial and failure. Early crystallographic studies suggested that finding blockers of RAS action would be a tough task with improbable success because the analysis of RAS structure did not show surface pockets where small molecule inhibitors could bind and block oncogenic signals . Due to Alternative strategies to delocalize RAS oncoproteins have also been developed recently. One of these involves the use of a novel inhibitor designed to disrupt the recently discovered interaction between KRAS and PDEδ, which is needed for KRAS placement in the inner side plasma membrane. This inhibitor, named Deltarasin was reported to prevent KRAS location to the plasma membrane and block pancreatic cancer cell proliferation . A diffeThe lack of effectiveness of FTIs in clinical trials, together with the absence of discernible pockets on the RAS surface allowing direct binding of inhibitor, led to the assumption that RAS could be “undruggable” and for many years the search for drugs against RAS-driven tumors focused on the inhibition of downstream signaling pathways . Most dohttps://www.clinicaltrials.gov, accessed on 5 April 2021). The initial notion that RAS was undruggable began to change with the discovery of pockets on the RAS surface that could be used to design new specific inhibitors capable of binding directly to the RAS oncoproteins. Based on structural and crystallographic studies showing that HRAS proteins cycle between two structural states  and that RAS proteins are unable to bind to effectors while in state 1, the existence of a groove in the surface of HRAS in state 1 was predicted that would be large enough to bind small molecule inhibitors potentially capable of blocking RAS-effector interaction . This seG12C that strongly and irreversibly bound to the mutated cysteine and prevented guanine nucleotide exchange, RAS activation and cancer cell growth [Clinical Trials.gov dedicated to analyzing the effectiveness and toxicity of KRASG12C inhibitors in solid tumors; especially lung cancers, where this mutation is frequent. Two of these inhibitors . At the(AMG 510  and MRTX(AMG 510 ) have re(AMG 510 .G12C inhibitors might be the first drugs targeting RAS directly that may reach FDA approval for clinical use.RAS research is entering its fifth decade, behind more than forty years of discoveries that have revealed a vast amount of information on these small GTPases. From the discovery of the viral oncogenes, and the isolation of their cellular counterparts, to the latest work on RAS inhibition and signaling, the knowledge on these proteins has allowed to design new strategies to target their role in cancer. Thus, new avenues to inhibit RAS translocation to the plasma membrane, to block downstream signaling pathways, or to directly inhibit their activity through small inhibitor binding, have come out in the last ten years, and covalent KRASDespite these recent advances, there is still a long way to go to effectively block RAS signaling in cancer. KRASG12C mutations comprise only a small percentage of RAS alterations in cancer and new drugs specifically targeting other RAS mutations are needed. Furthermore, resistance to drugs targeting RAS will likely limit their efficacy in the clinic , and effAnother relevant consideration for therapy of RAS-driven cancer is the long-known ability of RAS oncogenes to cause accumulation of genomic/chromosomal instability that critically contributes to progression through the evolutionary phases of tumorigenesis ,200. Thi


Since neurons have long neurites including axons, it is crucial for the axons to transport many intracellular substances such as proteins and mitochondria in order to maintain their morphology and function. In addition, mRNAs have also been shown to be transported within axons. RNA-binding proteins form complexes with mRNAs, and regulate transport of the mRNAs to axons, as well as locally translate them into proteins. Local translation of mRNAs actively occurs during the development and damage of neurons, and plays an important role in axon elongation, regeneration, and synapse formation. In recent years, it has been reported that impaired axonal transport and local translation of mRNAs may be involved in the pathogenesis of some neurodegenerative diseases. In this review, we discuss the significance of mRNA axonal transport and their local translation in amyotrophic lateral sclerosis/frontotemporal dementia, spinal muscular atrophy, Alzheimer’s disease, and fragile X syndrome. Neurodegenerative diseases chronically affect neurons in a specific network of the central or peripheral nervous system. Aging is one of the risk factors for the occurrence of these diseases, and the number of patients is increasing year by year with the increasing elderly population . The detC9orf72 gene  and frontotemporal dementia (FTD), changes in the localization of transactive responsive-DNA binding protein 43 (TDP-43)  and fusef72 gene . Alzheimf72 gene .SMN1) gene causes loss of functional SMN protein, resulting in damage of motor neurons (FMR1) gene reduces fragile X mental retardation protein (FMRP) production, resulting in autism spectrum-like symptoms , deletion or missense mutations of the survival of motor neuron 1 , an RNA granule-associated phosphoinositide-binding protein . MitochoIt is controversial which is more critical for triggering neuronal cell death in neurodegenerative diseases: changes in neuronal cell bodies (neuronopathy) or changes in axons (axonopathy). Nonetheless, there are many reports that indicate changes in axons or synapses occur during the early stage of the diseases. In ALS and SMA, morphological and functional abnormalities of the neuromuscular junctions between lower motor neurons and skeletal muscles, are detected in model mice that have not yet developed motor symptoms, as well as in patients with mild symptoms . In AlzhAs mentioned above, local translation in axons plays an important role in maintaining a healthy state of neurons. Therefore, it is presumed that if the local translation is disrupted, the function and viability of neurons will be impaired. In fact, several neurodegenerative diseases have been suggested to impair the local translation function, and are considered to be involved in the pathophysiology of various neurodegenerative disease models. In the following, we review dysfunctions of local translation in some typical neurodegenerative diseases.TARDBP), FUS, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), hnRNPA2/B1. As a frequent mutation in ALS/FTD, abnormal expansion of the GGGGCC repeat sequence in the untranslated region of the C9orf72 gene is also observed  site required for nuclear localization of FUS. These mutations are thought to be involved in diseases by changing the intracellular localization and enhancing aggregation of the proteins. Mutant TDP-43 and FUS show disturbance of stress granule dynamics  and microtubule associated protein 1B (MAP1B) mRNAs are reported to be transported to axons by TDP-43  gene have been identified in juvenile FTD  assembles small nuclear ribonucleoproteins (snRNP)  and formACTB) mRNA is reduced in axons of SMN-deficient motor neurons  mRNA is also transported and translated in axons under the control of SMN  is highly expressed in developing spinal motor neurons, and promotes axon branching and neuromuscular junction formation. CPG15 mRNA is transported to axons with the assistance of SMN and translated locally. Overexpression of CPG15 mRNA rescues SMN deficiency motor axon defects, indicating that localization of CPG15 mRNA in axons is an important determinant of axon architecture  binds to both  and SMN . Further15 mRNAs . These RDecreased expression of SMN has been shown to result in reduced overall translation in growth cones . This ma1–42, has the most potent aggregating property. Mutations in APP, presenilin 1 (PSEN1) and PSEN2 genes have been identified in familial AD patients, and these mutations are responsible for increased processing of Aβ1–42  is produced by processing from amyloid precursor protein (APP) and extracellularly deposited as amyloid plaques. These plaques and hyperphosphorylated tau which is characterized by intracellular accumulation as neurofibrillary tangles are hallmarks of the disease . One of f Aβ1–42 .In vitro, Aβ reduces BDNF signaling by inhibiting the proteolytic production of BDNF from pro-BDNF , a key molecule of the unfolded protein response pathway, thereby causing neurodegeneration. ATF4 mRNA and protein are both increased in axons of brain lesions in patients with AD, supporting the notion obtained from cultured neuron experiments.On the other hand, local treatment of Aβin axons . Among tMAPT mRNA, change the isoform expression pattern of MAPT mRNA and protein, increase the formation of neuronal RNA granules, and promote axonal elongation  domain and an arginine–glycine–glycine repeat (RGG) domain, which function to suppress the overall translation of proteins via binding to the RGG domain to G-quadruplexes of mRNAs , or bindRHOA mRNA in distal axons, which causes collapse of growth cones  mRNA into axons, and regulates their local translation via microRNA to act on axon elongation (The mechanism of translation suppression by FMRP has been vigorously investigated primarily in dendrites, specifically in postsynaptic spines . Howeverth cones . It has ongation .TUBA4A and NEFH are listed as the causative genes of some ALS cases (As we overviewed here, different RNA-binding or RNA-assembly protein abnormalities are reported to be associated with neurodegenerative disorders, and the corresponding target mRNAs have been identified in each of the proteins. Notably, many of the target mRNAs are cytoskeleton-related protein transcripts that reside in growth cones and synapses. These data suggest that axonal transport and local translation of these mRNAs are important for the morphological and functional formation and maintenance of growth cones and synapses. Dysfunction of the transport and translation of these mRNAs causes degeneration of axon terminals, which disconnects the neural network to cause symptoms of each disease . ImpairmLS cases , which aThe integrity of ribosomes in axons to maintain local protein synthesis capability may also be a critical factor for maintaining neuronal health. As we discussed above, RNA-binding and RNA-assembling proteins can regulate transport and translation of many different mRNAs in axons, rather than limited specific targets. In addition to our finding that TDP-43 targets translation-related mRNAs for axonal transport, change of overall translation efficiency is detected in axons as a result of TDP-43, FUS, and SMN dysfunction . ProteinAxonal transport and local translation of mRNAs play an important role in maintaining the survival of neurons, and their failure to do so is greatly involved in the development of many neurodegenerative diseases. By improving the function of mRNA metabolism in axons, it will be possible to develop new therapeutic strategies for these neurodegenerative diseases.SN and TA wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Several endogenous and exogenous factors interact to influence stroke occurrence, in turn contributing to discernable daily distribution patterns in the frequency and severity of cerebrovascular events. Specifically, strokes that occur during the morning tend to be more severe and are associated with elevated diastolic blood pressure, increased hospital stay, and worse outcomes, including mortality, compared to strokes that occur later in the day. Furthermore, disrupted circadian rhythms are linked to higher risk for stroke and play a role in stroke outcome. In this review, we discuss the interrelation among core clock genes and several factors contributing to ischemic outcomes, sources of disrupted circadian rhythms, the implications of disrupted circadian rhythms in foundational stroke scientific literature, followed by a review of clinical implications. In addition to highlighting the distinct daily pattern of onset, several aspects of physiology including immune response, endothelial/vascular and blood brain barrier function, and fibrinolysis are under circadian clock regulation; disrupted core clock gene expression patterns can adversely affect these physiological processes, leading to a prothrombotic state. Lastly, we discuss how the timing of ischemic onset increases morning resistance to thrombolytic therapy and the risk of hemorrhagic transformation. Stroke, caused by a sudden interruption to the blood supply of the brain, has a wide range of detrimental consequences that have contributed to its recognition as the leading cause of disability and second most common cause of death worldwide. It has serious social and economic consequences for both the individual and society. Ischemic strokes account for the majority of all strokes (87%), with intracerebral hemorrhages and subarachnoid hemorrhages being far less common , Brain and Muscle ARNT-like Protein 1 , Period (Per), and Cytochrome (Cry). Clock and Bmal1 encode for proteins containing bHlh-PAS domains, which form the positive arm of the feedback circuit by heterodimerizing to initiate transcription through binding to E-boxes (5′-CACGTG-3′) and (5′-CACGTT-3′) in the promoter of target genes. In turn, Per and Cry dimerize, acting as the negative limb of the feedback loop to inhibit Clock:Bmal1 transcriptional activity for the cycle to repeat, and degradation of Per and Cry proteins are responsible for terminating repression and restarting transcription. A second transcriptional feedback loop is initiated by the Clock:Bmal1 dimer which involves E-box mediated transcription of orphan nuclear-receptor genes Rev-Erbα/β and RORα/β  that depolarize and project through a monosynaptic neuronal pathway known as the retinohypothalamic tract (RHT) to the suprachiasmatic nucleus (SCN). The SCN serves as the master circadian clock in mammals and coordinates internal circadian synchronization; it sits at the top of a hierarchy of clocks of various tissue and cell types that display independent circadian gene expression patterns that are entrained by neural and humoral signals from the SCN . These nPer paralogs function as the state variable such that variations in the levels of proteins account for the phase of the clock  in Per promoters , with that statistic jumping up to 99% in the United States and European populations  and adrenalin, platelet adhesiveness was measured using blood samples from healthy subjects  over six timepoints ; prothrombin peaked at 1600 h, suggesting a transient morning hypercoagulable state  mice; euglobulin clot lysis time was reduced in Clock mutants and significantly increased in Cry1/2-deficient mice without time-of-day differences (0900 and 2100 h). Further characterization showed reduced PAI-1 fluctuation in Clock mutant mice while Cry1/2-deficient mice held similar levels at both 0900 and 2100 h  mice display progressing prothrombotic state characterized by reduced prothrombin times, increased platelet count, a reduction in nitric oxide/thrombomodulin expression from endothelial cells  , suggesting a significant role for Bmal1 in pericyte regulation for blood-brain barrier maintenance (Evidence of an endogenous circadian rhythm in blood brain barrier permeability has been characterized in Drosophila where rhodamine B and daunorubicin concentrations were significantly increased in the brain during the rest phase (ZT 12) compared to their active phase through increased efflux activity via pgp-like transporters during the day . This os period) . Further period) . Other antenance .Bmal1 knockout mice display lower blood pressure and abolished rhythmicity  followed by mid-morning peaks and decreases during the evening (inactive phase) . Additiothmicity , whereasosterone  and sodiosterone , both ofchannels , while dchannels . Furtherd injury .Nitric oxide (NO) and redox signaling involved in vascular contraction is another process that displays robust circadian oscillations involved in the vasoconstriction process. In the context of cerebral ischemia, NO synthases have both neuroprotective and damaging effects during brain hypoxia. Inducible NOS (NOS2) and neuronal NOS (NOS1) are involved in neurotoxicity, leading to excitotoxicity cascades, inflammation, apoptosis, and deterioration to the primary brain injury through the release of free radicals and production of nitrates, which directly damage mitochondrial enzymes and genetic material . EndotheComponents of the immune system are critical in pathophysiology of cerebral ischemia and are also regulated by the circadian clock. Acute cerebral hypoxia caused by thrombosis during ischemia triggers inflammatory cascades, and immune activation can cause secondary damage within the penumbra of the ischemic infarct . MoleculIn addition to the highly organized and regulated immune system, there are specific immune cell populations that exist in the CNS that are vital in maintaining normal brain homeostasis and function. Glial cells including microglia and astrocytes are non-neuronal populations in the central nervous system that function to maintain homeostasis, provide support, and protection, which are both involved in the innate and adaptive immune system . InflammBmal1 expression  along with decreased Nrd1, Dbp, Il1 β, and Nox2 expression and increased Gsr and Lpl expression, indicating that Bmal1 regulates immune response and cellular metabolism in microglia  revealed a 49% increase of occurrence between 0600 and 1200 h in all types of strokes, and 79% increase over normalized risk compared to the other 18 h of the day , with thTogether, the literature supports the notion that a time of day variation in stroke outcome exists, and that patients who experience stroke-like symptoms during the morning, or onset of the active period, have an increased risk for poor outcome. The data also suggest that it likewise could be possible to optimize therapeutic strategies based on the biological time that the onset of stroke occurs to improve survival rates. Time of day and circadian rhythms are both important variables in ischemia, and few clinical studies have considered these variables as biological factors. In this review we highlight the existence of circadian variations in ischemia and efficacy of thrombolytic intervention for clinicians, physicians, and pharmacists with the aim that time of day be recorded and reported as a biological variable in future clinical studies .Other aspects of temporal organization influence and alter stroke recovery. For example, exposure to artificial lighting from hospital recovery units is a major issue . Indeed,Bmal1, Per1, Clock, AKT, Erk-1/2, mTOR, S6, and BAD , foundational science research has examined the role of circadian disruption by light at night in stroke outcome. Studies evaluating the impact of ecologically relevant levels of dLAN (5 lux) have reported that stroke lesion size was significantly larger in dLAN mice compared to mice housed in dark nights after 24 h . FurtherBmal1, has also resulted in increased reactive oxygen species, which is critical during mediation/exacerbation of damage during reperfusion in ischemia , a serine protease which is responsible for cleaving plasminogen to plasmin . Plasminnd Bmal2 . In vivoor PAI-1 .Clock/Bmal1  involved in vascular matrix remodeling, specifically, MMP-2 and MMP-9 are upregulated after tPA administration in focal cerebral ischemia ; MMP-9 cck/Bmal1 . Thus, LIn the context of clinical studies, a small population study of 476 non-lacunar strokes treated with an IV infusion of tPA was interested in identifying possible circadian variation with thrombolytic intervention; patients who received IV thrombolysis at morning (0600–1200) and afternoon (1200–1800 h) time points exhibited less improvement on the NIH Stroke Scale compared to other time points. Furthermore, hemorrhagic transformation was also lower in frequency for patients who started IV thrombolysis between 1200 and 1800 h compared to those who received treatment between 0000 and 0600 h . TogetheStroke requires a rapid diagnosis and quick intervention because of the limited time for thrombolytic intervention, highlighting the importance of timing for this disease. Biological timing and clock gene expression play important roles in the cerebrovascular and cardiovascular systems. Basic foundational research has clearly demonstrated time of day variation in function, and the detrimental consequences of disruptions to circadian rhythms and their impact on contributing to a prothrombotic state. In addition to the emphasized morning circadian distribution pattern of stroke frequency, clinical studies support improved functional outcome and recovery in patients who receive thrombolytic therapy during the evening; the morning hypofibrinolytic and hypercoagulable state, in combination with increased PAI-1 levels, may contribute to less effective tPA treatment in the morning. Furthermore, patients have significantly lower neurological improvement rates when tPA is administered in the morning compared to the evening, as well as increased hemorrhagic transformation post thrombolytic intervention during morning time points. Thus, optimizing thrombolytic treatment based on time of day onset and the reduction of exposure to exogenous environmental circadian disruptors, such as exposure to lighting at night, has the potential to reduce secondary damage during stroke recovery and improve functional outcome. The intricate relationship among circadian rhythms, disruptions, and the stroke outcome, emphasizes the importance of chronopharmacological research.JL, JW, AD, and RN designed, wrote, and edited the manuscript. JL designed the figures. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The development of cancer treatment methods is constantly changing. For common cancers, our treatment methods are still based on conventional treatment methods, such as chemotherapy, radiotherapy, and targeted drug therapy. Nevertheless, the emergence of tumor resistance has a negative impact on treatment. Regulated cell death is a gene-regulated mode of programmed cell death. After receiving specific signal transduction, cells change their physical and chemical properties and the extracellular microenvironment, resulting in structural destruction and decomposition. As research accumulates, we now know that by precisely inducing specific cell death patterns, we can treat cancer with less collateral damage than other treatments. Many newly discovered types of RCD are thought to be useful for cancer treatment. However, some experimental results suggest that some RCDs are not sensitive to cancer cell death, and some may even promote cancer progression. This review summarizes the discovered types of RCDs, reviews their clinical efficacy in cancer treatment, explores their anticancer mechanisms, and discusses the feasibility of some newly discovered RCDs for cancer treatment in combination with the immune and tumor microenvironment. With the increase in the incidence of various types of cancer such as breast, kidney, and lung cancers, cancer therapy has always been the focus of clinical development . With thBased on the functional differences, there are two main types of cell death: ACD and RCD . ACD is Since the discovery of apoptosis in 1972 , 21, resApoptosis is an active programmed cell death caused by gene regulation that maintains homeostasis in the internal environment. The apoptosis activation pathway is diverse, including intrinsic and extrinsic pathways among others. These pathways will eventually promote the cysteinyl aspartate specific proteinase (caspase) cascade reaction; thus, apoptosis depends on the caspase family . The extIn normal cells, detection of irreversible DNA damage can induce apoptosis. However, cells having an overexpression of the apoptosis inhibitor Bcl-2  or p53 dApoptosis has conventionally been considered the only form of RCD, while necrosis has been considered a type of accidental death that is not regulated by molecular events. This misconception was later amended when necroptosis was discovered. It is a type of RCD resembling apoptosis in mechanism and necrosis in appearance . NecroptApoptosis and necroptosis, the first two forms of RCD discovered, have been playing an active role in cancer. The development of various new small-molecule drugs is aimed at activating the apoptotic pathway of cancer cells, thereby promoting cell death. The current mainstream direction points to the gene-level regulation of cellular apoptosis and necroptosis, and inhibiting or overexpressing the expression levels of related genes promotes the occurrence of cellular apoptosis and necroptosis. Not only these two RCDs, but more newly discovered RCD types have also been confirmed to be applicable in cancer treatment.Anoikis was first discovered in 1993 and explains the susceptibility of outlier cells to death , 62. ApoCancer cells acquire resistance against anoikis by regulating integrins and activating the EMT. Integrins play a key role in regulating cell contact with the ECM . IntegriAutophagy, a type of cell death that is completely varies from apoptosis, has been recognized to have a dual role where it can either inhibit or promote cancer metastasis . The antin vitro and in vivo trials are supporting this treatment strategy. Studies have shown that carnitine palmityl transferase 1A (CPT1A)-mediated fatty acid oxidation (FAO) can help colon cancer cells resist anoikis and thus promote colon cancer metastasis. Researchers have identified CPT1A as a potential target for colon cancer treatment . Bioinformatics analysis showed that CDO1 promoter methylation was significantly correlated with poor prognosis of ccRCC patients, suggesting that CDO1 promoter methylation may be a new prognostic molecular marker of ccRCC . StudiesAccording to the current research, the development of anticancer drugs based on ferroptosis mainly focuses on two aspects; system Xc and GPX4. The survival and growth of cancer cells strongly depend on the transport activity of system Xc-, making system Xc- a potential target for anticancer drug development.Systemic Xc- inhibitors can inhibit cystine uptake and interfere with cellular mechanisms that control protein folding, induce cellular stress, and thus lead to ferroptosis . ErastinSorafenib is a clinically approved multikinase inhibitor for the treatment of advanced cancers, such as renal and liver cancers , 135. ReIt is worth noting that some cancer cells can bypass system Xc- and synthesize cysteine through the transsulfation pathway. This suggests that inhibition of system Xc- therapy is not suitable for all cancers. GPX4 is a central regulator of iron function, and its inactivation leads to iron function loss, even at normal cysteine and GSH levels .RSL3 targets enzymes with nucleophilic sites  and inactivates GPX4 directly by alkylation of selenocysteine . One of FIN56 is a ferroptosis inducer derived from CIL56. Compared with CIL56, FIN56 has a higher efficiency and specificity in inducing ferroptosis. FIN56 promotes GPX4 degradation, which requires the enzymatic activity of acetyl-CoA carboxylase (ACC) and simultaneously binds and activates squalene synthetase (SQS), leading to the depletion of the endogenous antioxidant CoQ10 and enhanced cell sensitivity to fin56-induced ferroptosis . Sun et Since its discovery in 2012, ferroptosis has been widely studied as a therapeutic approach for the treatment of cancer; thus, its anticancer mechanism is worth further exploration.2+) signaling pathway or DNA modification  can also induce PARP1 activation  overactivation. PARP is a multifunctional post-translational modification enzyme that is present in most eukaryotic cells. It is activated by the recognition of structurally damaged DNA fragments and then performs DNA repair . PARP prtivation , 149 , they change from non-immunogenic to immunogenic and mediate anti-tumor immune response after tumor cell death. This phenomenon is known as immunogenic cell death (ICD) . The claWhen ICD occurs, HMGB1 is released from cells. The release of HMGB1 involves in it crossing the nuclear membrane and the plasma membrane to complete the transfer of HMGB1 from the nucleus to the cytoplasm and finally to the extracellular space. Extracellular HMGB1 binds to PRRs expressed on the surface of bone marrow cells, advanced glycosylation end-product-specific receptor (AGER or RAGE), and toll-like receptor 4 (TLR4), which activate corresponding signaling pathways and promote immune response . HMGB1 hWhen ICD occurs in tumor cells, CRT is exposed to the cell membrane surface and acts as an “Eat-ME” signal. This promotes dendritic cells (DCs) to engulf dead or dying tumor cells and promotes the maturation and function of DCs . Under svia the ANNexin channel. ICD is activated by the release of a “Find-me” signal via the purinergic receptor P2RY2, which is picked up by DC progenitors and macrophages. ATP released from the cell is also involved in mediating the development of proinflammatory cytokines, activating the formation of the casp1-dependent NLRP3 inflammasome and secretion of mature IL-1β and IL-18 , phenolic manganese dioxide nanoreactor, ferric iron, and polyethylene glycol polyphenol (MDP NPs) through metal phenol coordination assembly  , 194. BrNET release begins with the activation of neutrophils, where neutrophil surface receptors bind to ligands. Studies have shown that neutrophils lacking surface receptors do not develop NETosis . G proteThe role of neutrophils in tumor progression is controversial because neutrophils have both pro-tumor and anti-tumor properties . The neuMany of the above findings suggest that research on tumor-induced NETosis should focus on targeting NETs that may benefit cancer patients. As research progresses, NETs have shifted from being initially considered a defense against serious infectious diseases to negatively impacting the body during cancer by promoting deadly processes such as thrombosis as well as systemic inflammation and cancer recurrence. With this in mind, NETs could provide excellent targets for future anticancer therapies.Cells can quickly disintegrate and die when exposed to extremely harsh environmental conditions, which is called accidental cell death (ACD). Minor exogenous or endogenous disturbances promote adaptive stress, thus restoring intracellular homeostasis. When the stress response fails to restore homeostasis, one or more signaling cascades are activated in the cell, contributing to the regulation of cell death.There are many types of RCDSs with different mechanisms . Dalian Young Science and Technology Star from GW .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Grem1 has an increasingly recognised pleiotrophic role in a number of organ systems and is implicated across a wide range of disease states. Although the importance of Grem1 in intestinal stem cell regulation has been well described, a putative function in haematopoietic niche maintenance is novel and requires further exploration. Moreover, the complex and context‐specific regulation of Grem1, among a host of functionally convergent but structurally disparate BMP antagonists, warrants further research as we learn more about the pathogenic consequences of deranged expression of this small, but important, protein. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Highly conserved, complex and interacting morphogen signalling pathways regulate adult stem cells and control cell fate determination across numerous different organs. In homeostasis, the bone morphogenetic protein (BMP) pathway predominantly promotes cell differentiation. Localised expression of ligand sequestering BMP antagonists, such as Gremlin 1 (Grem1), necessarily restricts BMP activity within the stem cell niche and facilitate stemness and self‐renewal. In a new paper, Rowan, Jahns  Adult stem cells are critical to support tissue homeostasis, turnover, repair and regeneration, but cell fate determination in organ systems must be strictly regulated to prevent the unwanted development of neoplasia. A network of interacting morphogen signalling pathways, conserved across species, has evolved to regulate stem cell function. Many of these pathways generate concentration gradients through intercompartmental crosstalk, with differential expression of ligands, receptors and antagonists between different cellular components of the stem cell niche. Cell fate determination is regulated by position within these gradients. This multilevel signalling control and interactive pathway crosstalk permits amplification or attenuation of signal in a context‐dependent manner. Moreover, the dependence on intercompartmental interaction builds in capacity for functional compensation and buffering, preventing reliance on a single cell type for morphogenic control of epithelial cell fate. Unravelling the complexity of these networks, and identifying the key players within each tissue context, is critical to understanding cell fate control in homeostasis and its disruption in disease.et al [The Journal of Pathology used the ubiquitous ROSA26‐CreERT2 recombinase, activated by oral tamoxifen, to examine the systemic impact of Grem1 deletion on organ homeostasis in adult mice. Acute transgenic Grem1 knockout results in the rapid development of a severe enteropathy consistent with loss of proliferating stem/progenitor cells. These findings are consistent with the hypothesised role of this BMP antagonist as a key regulator of intestinal stem cell activity [et al also demonstrated that ubiquitous knockout of Grem1 caused the surprising and unexpected additional finding of a hypocellular bone marrow, reminiscent of aplastic anaemia [The bone morphogenetic protein (BMP) pathway is one of these critical morphogens, with stromal cell‐derived ligand activity promoting epithelial cell differentiation and apoptosis in multiple tissues. Thus, BMP activity must be restricted within the stem cell niche for maintenance of stemness, and this is achieved by the localised expression of a number of secreted ligand‐sequestering BMP antagonists. In the intestine, the sub‐crypt myofibroblast cells of the muscularis mucosae are the source of numerous secreted BMP antagonists, including Gremlin 1 (GREM1), Gremlin 2 (GREM2) and Chordin‐like‐2 (CHRDL2). The mechanistic effect of Grem1 is best understood predominantly through the pathological effect of aberrant epithelial expression, which promotes excessive stem/progenitor cell activity in the rare familial hereditary mixed polyposis syndrome [activity . HoweverGrem1, a key antagonist of BMP2/4 and 7 is of great interest. Grem1 has been shown to regulate bone marrow MSCs [Gradients of BMP signalling have long been known to regulate the proliferation and differentiation of haematopoietic stem cells (HSCs) in the developing embryo , and therow MSCs , but is et al together with accumulating literature evidence implies a critical role for Grem1 in embryonic development, homeostatic adult tissue stem cell regulation, regeneration and neoplasia, and indicates a key role for this antagonist in multiorgan BMP pathway regulation.This paper , intimatet al [Grem1 expressing cells [Grem1 was deleted using a different ubiquitous Cre recombinase (CAGG‐CreERT2) [As the pathological implications of disruption of Grem1 expression emerge, this paper highlights some key residual research questions. Rowan, Jahns et al  report ang cells . HoweverCreERT2) . The difGREM1 gene, but patients present with a colonic neoplastic disease phenotype. This is caused by a tissue compartment switch in expression from intestinal stromal to epithelial cells, with no apparent effect in other organs [Grem1 locus within an intron of the Formin gene [Based on the data presented here, Grem1 would appear to be a functionally important molecule in adult stem cell control, but the regulation of this gene also presents additional levels of complexity that need further research. Families with hereditary mixed polyposis syndrome have a germline 30 kb duplication on chromosome 15, upstream of the coding region of the min gene . Thus, iFrom this work and the increasing interest in the field, it is becoming evident that Grem1 plays an important role in modulating BMP signalling across multiple tissues and that disruption of this intercompartmental signalling pathway contributes to disease states in numerous organs. However, the more we learn, the more we need to understand through further research before we can realise the potential therapeutic benefits of manipulation of Grem1 signalling across a range of disease states.SG and SJL wrote the manuscript.


Pulmonary arterial hypertension (PAH) is characterised by an increased pressure in the pulmonary arterial circulation, resulting in the elevation of pulmonary vascular resistance. Pulmonary endothelial dysfunction and inflammation, triggered by shear stress and hypoxia, constitute the hallmarks of pulmonary vasculopathy by promoting endothelial and smooth muscle cells proliferation, vasoconstriction, and thrombosis. While research was predominantly focused on pulmonary vasculature, the investigation of peripheral endothelial damage in different vascular beds has attracted the interest over the last years. As a result, effective non-invasive methods that can assess the endothelial function and the architectural integrity have been utilized for the evaluation of pulmonary and peripheral vasculature. Non-invasive plethysmography, pulmonary flow reserve, nailfold videocapillaroscopy, near-infrared spectroscopy, and imaging techniques such as magnetic resonance angiography and perfusion imaging coupled by a number of biomarkers can be used for the assessment of peripheral vascular function in PAH individuals. In this review, we summarise and critically approach the current evidence of more systemic derangement of vascular function in PAH defined by novel, non-invasive methods employed for functional and morphological assessment of endothelium and microcirculation. In fact, CTD-PAH is the second most common cause of PAH after idiopathic PAH, representing one of the leading causes of death in this population.2 Beyond common pathogenetic mechanisms with other forms of PAH such as thrombosis, vasoconstriction, endothelial, and smooth muscle cells proliferation, a more pronounced inflammatory and autoimmune component has been described in CTD-PAH.3 Infiltrating macrophages and lymphocytes, antinuclear antibodies, rheumatoid factor, and proinflammatory cytokines have been detected in the pulmonary vessels, as well as the serum of patients with CTD-PAH.5 The clinical evidence of such observations suggests that early administration of corticosteroids and immunosuppressants in SLE and MCTD patients could prevent irreversible pathologic changes in pulmonary vessels and improve functional status.6Pulmonary arterial hypertension (PAH) is haemodynamically defined by a mean pulmonary arterial pressure (mPAP)>20 mmHg at rest, a pulmonary capillary wedge pressure (PCWP)≤15 mmHg, and an increased pulmonary vascular resistance  according to the 67 The arterial wall is comprised of three cellular layers: the tunica adventitia, the tunica media, and the tunica intima. The region enclosed by the vessel wall is called the vessel lumen. Exposure to genetic, as well as environmental factors, such as hypoxia, induces vascular remodelling in PAH; namely, smooth muscle cell proliferation in the tunica media and enhancement of endothelial cell apoptosis in the tunica intima. Endothelial-like cells, when proliferated in a disorganized manner, form glomeruloid-like lesions called “plexiform lesions”.8 Tuder et al. demonstrated that these lesions are responsible for the expression of vascular endothelial growth factor, and the hypoxia inducible factor -1a.8 Data collected from 62 PAH patients indicated that plexiform lesions can be detected in the lungs of up to 90% of PAH patients.9 Three-dimensional reconstructive studies were able to show another onion-skin arrangement of endothelial- and/or smooth muscle cells, situated in proximity to plexiform lesions.10 This cellular accumulation was characterised as an “obliterative concentric lesion”, and such lesions appear to be completely restricted to the pulmonary vascular lumen.The endothelium, the inner cellular lining of blood and lymphatic vessels, plays a key role in the regulation of vascular tone, permeability, coagulation, inflammation, and vascular homeostasis in response to various stimuli.11 Over the last years the interest has shifted towards the investigation of more systemic peripheral involvement of macro- and micro-circulation in patients with PAH. On the top of validated methods assessing structural and functional parameters of vascular health including flow mediated dilation (FMD) and pulse wave analysis, a number of novel sophisticated modalities such as nailfold videocapillaroscopy (NVC) and near-infrared spectroscopy (NIRS) have emerged for the evaluation of peripheral microcirculation with promising implications in PAH.12Impairment of endothelial function in pulmonary circulation is the initial and fundamental event in the development and progression of PAH. In particular, dysregulated synthesis and release of vasodilator mediators such as nitric oxide (NO) and prostacyclin accompanied by overexpression of vasoconstrictors, such as endothelin-1 culminate in vascular changes typically observed in pulmonary vasculature.The current review provides a thorough report of the available data regarding systemic vascular injury, macrovascular dysfunction and microcirculation changes in peripheral vascular branches in patients with PAH, assessed by non-invasive techniques. The potential utility of biomarkers of endothelial dysfunction as a part of non-invasive evaluation of generalized vasculopathy in subjects with PAH is also briefly discussed.13 Original research papers and review articles registered until the end of December 2020 were selected to be included in this review. Publications not in English and data from ongoing research were excluded.A MEDLINE and EMBASE search was carried out according to published guidance on narrative reviews, using the following terms: pulmonary arterial hypertension, pulmonary hypertension, idiopathic pulmonary hypertension, connective tissue related-PAH, systemic sclerosis-related PAH, endothelial dysfunction, pulse wave velocity, flow mediated dilation, nailfold videocapillaroscopy, and near-infrared spectroscopy.14 Interestingly, the hyperaemic response was significantly correlated with indices of disease severity; namely New York Heart Association (NYHA) classification, mean pulmonary pressure, six-minute walking distance, and arterial oxygen desaturation upon exercise. The generalized vasculopathy in patients with idiopathic and SSc-related PAH was confirmed in a later study with a larger number of participants, which again demonstrated endothelial dysfunction in PAH individuals compared to controls, while a trend towards increased arterial stiffness in SSc subgroup was also noticed.15 Finally, Wolff et al. found that endothelial dysfunction in peripheral, non-pulmonary conduit vessels assessed by FMD in idiopathic PAH was correlated with acute pulmonary vasoreactivity defined as a positive pulmonary vascular response to inhaled iloprost during right heart catheterization (RHC).16 The later provides a link between systemic vascular injury and hemodynamic parameters supporting the hypothesis that at least to some extent, PAH can be perceived as a systemic vascular disorder affecting both the pulmonary and the peripheral vasculature.The presence of a more generalised peripheral impairment of endothelial homeostasis in PAH was originally determined by measurements of peripheral endothelium-dependent vasoreactivity using FMD, after brachial artery occlusion in individuals with idiopathic and SSc-related PAH, as well as patients with chronic thromboembolic pulmonary hypertension (CTEPH). Peled et al. reported abnormal endothelial function in all PAH groups compared to controls.17 The pulmonary flow reserve was determined by acetylcholine infusion into segmental pulmonary arteries using quantitative angiography and an intra-arterial Doppler flow wire in 17 children with IPAH. Endothelial function was then assessed by classifying the reactivity of the pulmonary to systemic arterial pressure ratio, short-term oxygen and intravenous epoprostenol or aerosolized iloprost administration. The results indicated that acetylcholine infusion can cast a light on the pathogenesis of IPAH providing valuable information regarding the prognosis of the disease in patients with impaired vasoreactivity.Early pulmonary vascular disease is often clinically silent, as initial pulmonary microvascular damage does not lead to significant changes in the pulmonary arterial pressure. Subsequently, there is an increasing interest in assessing the status of pulmonary microcirculation in early stages before advanced alterations occur. Zimmermann et al. attempted to determine the degree of pulmonary endothelial dysfunction in children with IPAH by using the pulmonary flow reserve method.18 Therefore, it becomes apparent that pulmonary flow shows promising results for the evaluation of endothelial activity by measuring vasoreactivity. However, this method is still in its infancy and further studies in humans are needed to validate these early findings.Another method for evaluating the pulmonary micro-circulation is based on the assessment of the peak hyperaemic flow using the thermodilution derived mean transit-time. Results from a study performed on baboons, in which peak hyperaemic flow was assessed using sensor-guidewires and adenosine/papaverine as hyperaemic agents, showed that these novel indices can detect progressive pulmonary microvascular degeneration and can contribute to the assessment of early pulmonary vascular disease.19 The most common parameters assessed are capillary density per linear millimetre, capillary dimensions (mainly capillary loop width), the presence of microhaemorrhages and capillary shape abnormalities.20 Given its ability to visualise different stages of peripheral vasculopathy, NVC is considered as a surrogate marker of SSc severity and progression, including vascular impediments such as PAH and/or visceral organ involvement.21 Similarly, in PAH, peripheral microangiopathy –assessed by NVC– may reflect the progression of pulmonary vasculopathy and provide a non-invasive, indirect tool for the evaluation of the disease itself.22 For example, a study including 12 SSc-related PAH patients and 12 SSc patients without PAH found a significant correlation between mPAP and worse NVC score, defined by the presence of enlarged, giant, ramified, or bushy capillaries, microhaemorrhages, and loss of capillaries.23 Abnormal capillaroscopic pattern has also been correlated with pulmonary vascular resistance in SSc-related PAH.24Nailfold videocapillaroscopy is a simple, non-invasive method used to recognize morphological and/or functional abnormalities in the peripheral microvasculature. It was initially developed for the diagnostic assessment of patients with Raynaud’s syndrome and is currently included in the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR) update of the classification criteria for SSc.25 This terminology, used to classify NVC abnormalities amongst SSc subjects, depicts distinct patterns visualised at different stages of the disease. For instance, “early” changes are characterised by the presence of few giant capillaries and microhaemorrhages, whereas “late” changes include progressive capillary loss and disorganized capillaries. This study indicated a strong correlation between NVC patterns and future severe vascular and lung involvement according to worsening scleroderma patterns. A recent meta-analysis confirmed the notable associations between SSc-PAH, capillary density and NVC pattern suggesting the potential integration of NVC into the current workout for the early detection of vascular injury in patients with CTDs.26Another study in 66 consecutive patients aimed to relate NVC scleroderma patterns classified into “normal”, “early”, “active” or “late” with organ involvement.27 Apart from the changes detected in SSc-PAH, they also demonstrated increased capillary width in patients with idiopathic PAH patients compared to healthy controls. The bidirectional relationship between peripheral microvascular remodelling and PAH was also examined by Hofstee et al., who described a decreased capillary density in both SSc-PAH and IPAH individuals, presenting such changes as an indicator of disease severity.28 Last but not least, recent reports including patients with idiopathic PAH and Eisenmenger syndrome have revealed abnormal capillaroscopic patterns across a wider spectrum of groups with pulmonary vascular disease.29 Finally, a recent study demonstrated significant NVC changes, such as reduction in capillary density and increased capillary dimensions in patients with IPAH and CTEPH. More than half of patients with IPAH presented microhaemorrhages on capillary nailfold, while shape abnormalities and capillary thrombi were detected at a greater extent in patients with CTEPH.30 This study is part of a larger prospective study focusing on the documentation of peripheral microangiopathy in patients with pre-capillary PH , as well as the association of qualitative and quantitative NVC parameters with clinical, haemodynamic, echocardiographic, biochemical and functional parameters of disease severity in PH patients .31In the context of PAH of other aetiologies, a number of studies have investigated NVC alterations in patients with idiopathic PAH. Corrado et al. studied 39 SSc patients (19 with PAH), 21 subjects with idiopathic PAH, and 20 healthy subjects.2), which is typically reduced in PAH patients.32 Spatially resolved NIRS provides a non-invasive and rapidly responsive method for assessing skeletal muscle oxygenation. This is accomplished by measuring absorption differences in the near-infrared spectrum of light between oxy- and deoxy-haemoglobin and myoglobin molecules in the microvasculature. The tissue oxygenation index (StO2) is adopted for the depiction of the dynamic balance between local tissue oxygen supply and utilization in all subjects.34 Since the contribution of myoglobin to the NIRS signal is not critical, StO2 is generally used as the ratio of oxygenated to total tissue haemoglobin concentration. A study by Panagiotou et al. correlated measurements of StO2 with SvO2 and venous oxygen saturation in the inferior vena cava during RHC to evaluate the use of NIRS in PAH.35 For this purpose, 25 PAH patients were assessed at rest, whereas 10 of those subjects were also examined during exercise. Results showed that skeletal muscle StO2 in PAH subjects correlated considerably with SvO2 under resting and exercise conditions and furthermore, it is possible that this could indicate disease severity. Therefore, NIRS could serve as a non-invasive tool for the qualitative evaluation of the dynamic balance between oxygen supply and demand in PAH as well as for the assessment of disease progression and severity. Inspired by the work of Panagiotou et al., NIRS was used to confirm the significant contribution of peripheral muscle alterations to the exercise intolerance in PAH patients.36 Finally, Dimopoulos et al. assessed peripheral muscle microcirculation by utilising NIRS in 8 PAH patients, 8 matched healthy subjects (controls), and 16 patients with chronic heart failure.37 Tissue O2 saturation was measured on the thenar muscle as the haemoglobin saturation (%) in the microvasculature compartments. Subsequently, the 3-min brachial artery occlusion technique was applied before, during and after 15 min of breathing 100% O2. The oxygen consumption rate, the reactive hyperaemia time, and the time needed for tissue O2 saturation to reach its baseline value after the release of the occlusion were measured. PAH subjects presented a significant impairment of peripheral muscle microcirculation, decreased tissue O2 saturation (possibly attributed to hypoxemia), slower reactive hyperaemia time  and peripheral systemic vasoconstriction. Finally, acute hyperoxic breathing improved resting tissue O2 saturation and reduced the oxygen consumption rate and reactive hyperaemia time during reperfusion, probably due to increased oxidative stress and evoked vasoconstriction.38 These findings demonstrate an imbalance in oxygen supply and demand in peripheral muscles in patients with PAH, a phenomenon which may contribute to exercise intolerance in this population.The increased vascular resistance in PAH leads to a disturbance in oxygen delivery to the periphery and an impairment in tissue oxygenation. The balance between oxygen supply and demand is reflected in the index mixed venous oxygen saturation . For example, Oxygen-sensitive He-3 MRI allows for quantification of the alveolar partial pressure oxygen, which can serve as an important complement for comprehensive ventilation-perfusion MRI. However, it has yet to be approved for clinical use as an MRI contrast agent by the FDA mainly due to limited availability (it is a by-product of tritium decay and is therefore classified as a strategic product in the United States). In the case of PH, MR angiography and MR perfusion imaging allow an in-depth evaluation of the localisation of the chronic thromboembolic material (underlying cause of PH).41 Moreover, perfusion MRI can be quantitatively evaluated in order to assess the severity of the small vessel degeneration. Perfusion MRI is a sensitive surrogate for monitoring the disease, while structural imaging of the lung allows the exclusion of other parenchymal diseases. Lastly, measurements of blood flow and right heart structural modifications provide estimates for changes in pulmonary arterial pressure as well as indications of cardiac strain and associated valvular disease.42Shifting the focus from the visualisation of the larger vessels (macrocirculation) down to the segmental branches and the effect of the contrast agent during its passage through the arterioles and the capillary bed (microcirculation) paved the way to the development of perfusion magnetic resonance imaging (MRI).43 Quantitative pulmonary perfusion parameters such as pulmonary blood flow, mean transit time and pulmonary blood volume showed significant differences between normal volunteers and PAH patients. Thus, it is possible that three-dimensional ultrafast dynamic contrast-enhanced MR imaging can be used for the assessment of pulmonary perfusion parameters in the entire lung of both normotensive and hypertensive patients. Consequently, MRI techniques can be used for the visualization of the pulmonary vascular bed and for the assessment of pulmonary perfusion parameters but at this moment its sensitivity is limited due to lack of a highly available and effective contrast agent.A study conducted by Ohno et al. aimed at the quantitative assessment of regional pulmonary perfusion in the entire lung, using 3D ultrafast dynamic contrast-enhanced MRI in 40 volunteers .44 A potential endothelial cell marker is asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase. Impairment of the enzymatic activity of endothelial NO synthase leading to reduced bioavailability of NO is considered one of the main pathophysiologic mechanisms of PAH and is mainly mediated by ADMA. ADMA metabolism is dysregulated in PAH and high serum ADMA levels can be a useful biomarker for PAH progression.46 A study by Giannakoulas et al. also underlined the importance of ADMA in the assessment of pulmonary endothelial dysfunction associating it with changes in the expression and activity of connexin.47The detection and quantification of circulatory molecules that are correlated with endothelial degeneration and dysfunction are useful for the early detection and monitoring of PAH. Biomarkers can be roughly categorised according to their involvement in endothelial cell dysfunction, inflammation, epigenetics, cardiac function, oxidative stress, metabolism and extracellular matrix.48 The correlation between galectin-3 and PAH was studied in 15 patients with PAH who underwent echocardiography and a blood test to measure galectin-3 concentrations. The results showed an elevated galectin-3 in all PAH patients.49 Although these findings were encouraging, galectin-3 lacks the specificity to serve as a sole biomarker, since elevated concentrations can also be detected in patients diagnosed with renal failure, pulmonary and hepatic fibrosis.50Another inflammatory circulating molecule that could hold value as a biomarker is galectin-3. Galectin-3 functions as an endocrine and paracrine factor for the stimulation of macrophages, fibroblasts, and inflammatory cells. An increase in galectin-3 concentration has been found in patients with left heart failure where it seems to play an important role in cardiac fibrosis and remodeling.51 Moreover, they obtained lung tissue from the aforementioned patients, which they also examined for endoglin expression and microvascular endothelium changes. Expression of these molecules was significantly enhanced in both blood and tissue samples. Tiede et al. confirmed these findings in another study of 76 PAH patients (without however finding a connection between sVEGFR1 and haemodynamic parameters).52 sVEGFR1 is likely a modulator of chronic hypoxia-induced pulmonary and peripheral vascular remodelling, which mobilizes endothelial progenitor cells (EPCs). Diller et al. demonstrated that circulating EPCs were markedly reduced in Eisenmenger syndrome and IPAH compared to controls  and were also negatively associated with inflammatory biomarkers and asymmetric dimethylarginine.53 In IPAH, the number of circulating EPCs correlated with invasive hemodynamic parameters and treatment with the phosphodiesterase inhibitor sildenafil was associated with a dose-dependent rise in EPC numbers. In contrast to EPCs, circulating endothelial cells (CECs) are increased in PAH, since they are detached from pulmonary endothelium. Both in PAH associated with congenital heart disease (PAH-CHD) and IPAH, clinical deterioration was associated with increased CEC count, while treatment with PAH-targeted therapy was associated with a decrease.54 In addition, CEC count in peripheral blood was 10-fold higher in irreversible versus reversible PAH-CHD and controls.55Circulating factors that regulate angiogenesis are also examined for their role as biomarkers in PAH. Malhotra et al. investigated the concentrations of soluble endoglin, vascular endothelial growth factor receptor-1 (sVEGFR1), N-terminal brain natriuretic peptide and C-reactive protein in the peripheral blood of 97 PAH patients.56 Furthermore, research by Kumpers et al. demonstrated that high angiopoietin 2 blood concentration correlates with high pulmonary vascular resistance and is consequently an accurate predictor of survival.57 Lastly, Yang et al. detected high ghrelin concentrations in IPAH patients.58 Ghrelin is a hormone produced in the gastrointestinal tract responsible for regulating appetite. This hormone also proved to correlate positively with elevations of the pulmonary arterial systolic pressure, plasma N-terminal brain natriuretic peptide, endothelin-1 and nitric oxide levels.Another circulating molecule that was found to have a strong correlation with disease severity and mortality in PAH was the potent angiostatic factor, endostatin. Damico et al. discovered a direct association between high serum endostatin concentrations and poor functional status, decreased exercise tolerance, invasive hemodynamic variables and mortality.59 Sarrion et al. studied the role of various miRNAs as potential biomarkers of IPAH.60 Out of the miRNAs examined, miR-23a proved to be of special interest due to its association with pulmonary function.MicroRNAs (miRNAs) as biomarkers and coding genes are responsible for post-transcriptional regulation of gene expression as well as RNA silencing. Their serum concentration can be construed as a direct reflection of existing vascular pathology.61 Flow cytometry analyses of circulating lymphocytes were used to investigate the systemic mark of pulmonary lymphoid neogenesis. The results of this study demonstrated that IPAH lungs, compared to controls, contained perivascular ectopic (tertiary) lymphoid tissues, as well as B- and T-cell areas with high endothelial venules and dendritic cells. It remains to be seen whether the detection of such auto-antibodies and other lymphoid neogenesis factors, using flow cytometry, could serve as biomarkers for the early detection and diagnosis of IPAH, or if they serve as indicators of a poorer prognosis.The issue of pulmonary lymphoid neogenesis in IPAH was raised by Perros et al.2 saturation (possibly attributed to hypoxemia), slower reactive hyperaemia time  and peripheral systemic vasoconstriction. Additionally, MRI techniques can be used for the visualisation of the pulmonary vasculature and for the assessment of pulmonary perfusion parameters, but the availability of an effective contrast agent is, to date quite limited. Finally, circulating molecules, such as ADMA, galectin-3, ghrelin, MiRNAs and auto-antibodies can be detected and quantified in PAH patients and could serve as biomarkers for disease diagnosis, severity, and progression. While all aforementioned assessment methods are of great promise, further research and clinical studies are required in order to validate their role in an algorithm for a non-invasive detection and monitoring of systemic vascular changes in PAH.Endothelial dysfunction appears to be the trigger for microvascular degeneration and remodelling in PAH. These vascular changes are no longer considered organ-specific, but have been found to extend to the periphery as well. PAH can present itself as a severe complication of a pre-existing condition, as is the case in many patients with SSc and MCTD. Therefore, finding an available, cost-effective, and non-invasive technique for the evaluation of endothelial function in PAH and other high-risk patients would be of great value for early diagnosis as well as for continuous monitoring of disease progression. Plethysmography and fingertip tonometry for the assessment of endothelial function and arterial stiffness has shown promising results in SSc-PAH and IPAH patients. Furthermore, NVC makes it possible to detect changes in capillary density and dimensions as well as microangiopathy in the periphery directly correlated with disease severity. NIRS provided an explanation for exercise intolerance in PAH patients by demonstrating an imbalance in oxygen supply and demand in skeletal muscles. In more detail, findings in PAH subjects using NIRS include significant impairment of peripheral muscle microcirculation, decreased tissue O


PTEN, TP53, RB1, CDK12, and DNA repair, or specific activation of regulatory pathways, such as ETS or MYC, may also facilitate immune evasion of the host response against cancer. This review presents an update of recent discoveries about the role that the common somatic mutations can play in changing the tumor microenvironment and immune response against prostate cancer. We describe how detailed molecular genetic analyses of the tumor microenvironment of prostate cancer using mouse models and human tumors are providing new insights into the cell types and pathways mediating immune responses. These analyses are helping researchers to design drug combinations that are more likely to target the molecular and immunological pathways that underlie treatment failure.Immunotherapy has improved patient survival in many types of cancer, but for prostate cancer, initial results with immunotherapy have been disappointing. Prostate cancer is considered an immunologically excluded or cold tumor, unable to generate an effective T-cell response against cancer cells. However, a small but significant percentage of patients do respond to immunotherapy, suggesting that some specific molecular subtypes of this tumor may have a better response to checkpoint inhibitors. Recent findings suggest that, in addition to their function as cancer genes, somatic mutations of  Previous approaches to cancer therapy were based mostly on how to control clonal proliferation of aggressive tumor cells. Recently, translational researchers have focused on the role of the anti-cancer immune system in the design of new therapies to improve the response and overall survival. Thus, an important new hallmark of cancer is understanding how to stop tumors bypass the host’s protective immune destruction mechanisms .CDK12 biallelic mutations responding to this therapy , atezolizumab [n = 1], ipilimumab + nivolumab [n = 1], tremelimumab + durvalumab [n = 1] and atezolizumab [n = 1]); 11/19 (59%) had a decline in PSA level, with 2 patients (11%) having 100% PSA decline. Therefore, tumors harboring CDK12 loss of function seem to be more aggressive and invasive than other PCa subtypes and responded to ICB. However, there were some patients from this subgroup who were non-responsive to ICB treatment despite harboring CDK12 alterations.There is currently considerable interest surrounding whether the combination of PD-1 and CTLA-4 inhibitors would be more efficacious in these genomically selected patients with mutation ,133. Wu mutation . In a re4 months . In a re12 mCRPC . In thisAnother class of genetic alteration that has drawn attention for combinatorial use of immunotherapy are the mutations in the DNA repair pathway genes. Combinations of PARP inhibitors in tumors with mutations in homologous recombination genes may result in loss of compensatory DNA repair mechanisms, leading to death of cancer cell due to unrepairable DNA damage ,136.There is recent interest in new classes of immunotherapeutic drugs that take advantage of interactions of the innate immune system between cancer cells and macrophages through the CD47 and the signal regulatory protein alpha protein (SIRPα) ,138. CD4Improved understanding of the tumor biology of immune response has been greatly facilitated by recent technological advances in the analysis of the PCa microenvironment. Detailed transcriptome characterization of tumor cells and the non-cancerous cells in the TME is providing important clues about the role of somatic mutations may play in modulating the crosstalk between cancer cells and the immune system. Studies of PCa using single-cell sequencing, spatial immunobiomarkers, and functional analyses in model systems are collectively providing novel insights into the composition, function, and location of immune cells and other non-cancerous cells within the TME. These ongoing studies in mouse models and human tumors are designed to investigate the best predictive somatic mutation biomarkers for immunotherapy in advanced PCa. Molecular profiling is increasingly guiding treatment decisions and sequential treatments of advanced cancers. The application of new immunobiomarker assays in PCa promises continuing improvements in immunotherapy success in future years.