Osteonecrosis of the femoral head is a progressive and debilitating condition with a wide variety of etiologies including trauma, steroid use, and alcohol intake. Diagnosis and staging are based on imaging including MRI at any stage and plain radiography in more advanced lesions. The only definitive treatment is total hip arthroplasty, although numerous treatments including disphosphonates and core decompression are used to delay the progression. Lack of satisfactory conservative measures suggests the need for additional research of osteonecrosis including large patient registries to further understand this condition. This review characterizes the current knowledge on etiology, pathophysiology, epidemiology, and clinical management of osteonecrosis, with an emphasis on recent developments.Osteonecrosis is a progressive disorder in which lack of sufficient blood supply leads to cell death, fracture, and collapse of the affected area. The condition is frequently associated with the femoral head, where progression can be debilitating and can ultimately necessitate total hip arthroplasty (THA). The etiology of osteonecrosis is complex with numerous contributing agents, most markedly trauma, steroid use, and alcohol. Treatment of osteonecrosis is controversial because no option has been overwhelmingly embraced, and little research has compared treatments. Researchers estimate that 20,000 new cases of osteonecrosis are diagnosed in the United States each year.1 Recent analysis has shown that although the number of THAs done for osteonecrosis has increased between 2001 and 2010 , the percentage of THAs done for osteonecrosis has decreased from 9.7% to 8.3%, likely because of the rapid increase in osteoarthritis necessitating THA.2The incidence of osteonecrosis in the United States has been estimated at ∼20000 to 30000 cases per year, affecting primarily young adults between the ages of 20 to 40 years.3Osteonecrosis occurs because of compromised blood flow or oxygen delivery to the bone, although the clinical presentation is a result of the repair process, rather than initial ischemia. In osteonecrosis, bone formation by osteoblasts is unable to match bone resorption by osteoclasts. This remodeling imbalance does not adequately replace the necrotic bone, leaving a region of structurally unsound bone tissues.4 disrupting blood flow and leading to osteocyte death. Estimates of occurrence of traumatic osteonecrosis of the femoral head vary depending on the injury type5; however, in meta-analysis of traumatic osteonecrosis, incidence has been found to be as high as 14.3%.6 The Garden classification categorizes femoral neck fractures and can be used to estimate the risk of osteonecrosis. Garden I (incomplete fracture) and Garden II (complete and nondisplaced) are considered stable and low risk, and can be repaired with internal fixation. Garden III  and Garden IV (complete and completely displaced) have much higher rates of osteonecrosis with internal fixation (16%), and arthroplasty should be considered.7 Intertrochanteric hip fractures result in a low risk of osteonecrosis, noted at 0.95% after a 1-year follow-up.8Trauma is the most common cause of osteonecrosis,10 The reasons for the sparing of one hip in the presence of a systemic risk factor are not well studied and may be because of subclinical presentation, differences in wear patterns between hips, underinvestigation of symptoms, or lack of coordinated follow-up.Atraumatic osteonecrosis encompasses a diverse array of causes. It is important to note that in atraumatic osteonecrosis, disease is frequently bilateral owing to systemic risk factors, with some estimates suggesting as high as 70% of the patients with unilateral osteonecrosis developing disease in the contralateral hip.12 Several potential mechanisms have been proposed for this association, including bone matrix and cartilage degeneration, induced stem cell abnormalities, changes in lipid metabolism, creation of fat emboli, altered coagulation, and changes in blood supply.12 Meta-analysis found up to 10 times increased risk of patients on high-dose corticosteroids, a doubling of risk for osteonecrosis when the cumulative dose exceeds 10 g, and a 0% increase in risk with each 10 mg increase of daily dose.13 Corticosteroids have also been implicated in osteoblast death and decreased osteoblast proliferation, impairing the ability to repair and replace necrotic lesions.11Steroid use is the second most common cause of osteonecrosis.14 It is hypothesized that increased generation of lipids increases the risk for fat emboli leading to vascular occlusion. In addition, increased serum lipids can cause packing of the marrow, increasing intraosseous pressure and decreasing blood flow.12 Alcohol may also contribute to osteocyte death.5 A study has also shown increased cortisol levels in patients with alcohol-induced osteonecrosis compared with idiopathic osteonecrosis control subjects, suggesting that alcohol-induced osteonecrosis may act through the steroid pathway.15 Previous estimates noted an 11 times higher risk of osteonecrosis in consumers of >400 mL of alcohol daily.16Alcohol is hypothesized to act through altered lipid metabolism and increased adipogenesis.4 One study of low-energy femoral neck fractures in the elderly found higher blood lipid abnormalities in those who developed osteonecrosis than those who did not.17 A study of patients with acute lymphoblastic leukemia (ALL) identified hyperlipidemia as a risk factor for developing osteonecrosis.18 A similar study found association with osteonecrosis development in patients with hyperlipidemia and systemic lupus erythematosus (SLE).19Hyperlipidemia is thought to decrease the blood supply to affected regions by increasing intraosseous pressure and producing fat emboli.20 Meta-analysis of SLE studies has identified numerous noncorticosteroid risk factors in SLE, notably renal involvement and central nervous system (CNS) disease.22 Mixed data suggest that the prothrombotic effects of antiphospholipid antibodies play a role in osteonecrosis development in SLE. Recent meta-analysis of childhood-onset SLE found notable osteonecrosis association, with estimates that 6 to 8.4% of the patients with childhood-onset SLE develop osteonecrosis,23 although most did not develop osteonecrosis until after puberty.21The association of SLE with osteonecrosis is related to frequent corticosteroid treatment; however, recent analysis has shown higher incidence of osteonecrosis in corticosteroid users with SLE than in corticosteroid users without SLE, suggesting synergistic effects.24 Precipitation of hemoglobin S in low-oxygen environments may lead to vaso-occlusion and ischemia of the bone, which is similar to the development of other vaso-occlusive injury in sickle cell disease.5 A recent study supports this theory, citing elevated hemoglobin levels as a risk factor for osteonecrosis in patients with sickle cell disease and suggesting that vaso-occlusion, high blood viscosity, hypoxia, and concurrent alpha-thalassemia contribute to osteonecrosis.25Studies of the association between sickle cell disease and osteonecrosis have identified 2 to 4.5 cases of osteonecrosis per 100 patients with sickle cell disease.26 Gaucher disease may act through a similar path to that of sickle cell disease, with Gaucher-affected cells obstructing the blood flow27 or by increasing intraosseous pressure because they accumulate in the fatty marrow.3 In addition, Gaucher cells can release osteoclast-activating cytokines which disrupt the balance of bone formation and resorption.26 Enzyme replacement can reduce or delay the symptoms of osteonecrosis28; however, a study has suggested that the bone marrow may serve as a “sanctuary site” for Gaucher cells, leaving a subset of patients vulnerable to osteonecrosis despite treatment.29A recent evaluation of the Gaucher Registry estimated the incidence of osteonecrosis at 30%.30 A recent study of divers with musculoskeletal decompression sickness found evidence of dysbaric osteonecrosis in 26% of the cases, although the study was limited by the relative rarity of this condition.31Decompression sickness–related osteonecrosis or dysbaric osteonecrosis occurs because of rapid decompression after an extended period in a hyperbaric environment. Rapid decompression forms bubbles in the bloodstream because dissolved nitrogen comes out of the solution. The high solubility of nitrogen in fatty tissues makes the marrow particularly susceptible. Multiple mechanisms have been proposed, including direct occlusion of blood flow to the marrow and the increase in intraosseous pressure reducing effective blood flow.32 The single largest factor identified in the development of osteonecrosis in patients with ALL is adolescence, suggesting an effect of ALL or its treatment on the growth and remodeling of the bone. It is also possible that the occurrence of this time of changing metabolism and growth magnifies susceptibility to osteonecrosis-causing damage from other factors.33 Older adults, who make up a small portion of those diagnosed with osteonecrosis, often undergo modified treatment regimens and have worse overall outcomes compared with their younger counterparts.34 A recent study of childhood leukemias found higher incidence of osteonecrosis in patients treated with hematopoietic stem cell transplant (HSCT) versus chemotherapy alone (6.8% versus 1.4%), suggesting that treatment methods influence osteonecrosis development.35 In addition, a review of treatment regimens identified increased cumulative dose of steroids as a risk factor for developing osteonecrosis in children with any hematologic malignancy.36 A review of treatment strategies suggested that the use of discontinuous steroid regimens may decrease the risk of osteonecrosis and nonsteroid chemotherapeutic agents such as methotrexate and asparaginase may contribute to the development of osteonecrosis.37 One trial of alternate week dexamethasone reduced the risk of osteonecrosis compared with continuous treatment in children with high-risk ALL.38Patients with ALL show an increased risk of osteonecrosis, with radiographic incidence reaching 71.8% in prospective studies.39A recent study suggests steroid-mediated development of osteonecrosis in transplant patients, finding cumulative steroid doses to be higher in renal transplant patients who developed osteonecrosis than in those who did not. The study also found that the incidence of symptomatic osteonecrosis decreased from 20% to less than 5% with the introduction of cyclosporine and a decrease in steroid usage.40 One recent study revealed a strong association between high-activity antiretroviral therapy and development of osteonecrosis, although the authors caution that the association does not imply a pathologic role.40 Other studies have found no association between osteonecrosis and antiretroviral therapy (ART), citing instead association with alcohol, hyperlipidemia,41 or low nadir CD4 counts,42 although the mechanism is not well understood.Multiple studies show a growing incidence of osteonecrosis in patients with HIV, showing nearly three times the risk of the general population.43 Elevated levels of osteoprotegerin and decreased expression of RANK/RANK ligand have been found in necrotic regions compared with healthy bone, suggesting a potential role of osteoclast-regulating genes.44 Factor V Leiden mutations and prothrombin mutations have been associated with patients with osteonecrosis in multiple studies,43 invoking a potential role of altered coagulation. Genome-wide association studies of selected populations have identified several loci of interest, including clusters of variants near glutamate receptor genes in patients with ALL,45 corticosteroid-induced osteonecrosis,33 and several loci of unknown significance, which may be related to coagulation pathways, lipid metabolism, or alcohol drinking behavior.46Although familial variants of osteonecrosis and some associated genes have been found, no single responsible gene has been identified. One gene candidate is a mutation in type II collagen, although no definitive causality has been established.47 This high rate of an unknown cause may be due to nonspecific early symptoms and indolent course, which prevent early diagnosis,9 as well as lack of standardized reporting and data collection, which may help to reveal little understood causes and connections.It is important to note that an estimated 20% to 40% of osteonecrosis cases are idiopathic.47 Diagnosis of osteonecrosis is primarily based on imaging, although examination and history are important to gather surrounding context and potential etiology.5 A plain radiograph is an appropriate first-line modality for identifying cases of osteonecrosis, with benefits including low cost, high availability, and adequate sensitivity for mid-stage and late-stage disease.48 Frontal and lateral “frog-leg” views are recommended for accuracy. In the case of early-stage disease, radiography may be insufficient to identify early or minimal changes. MRI is the benchmark for diagnosis of osteonecrosis because of its high sensitivity for early signs of onset. Supplemental imaging, including diffusion-weighted MRI49 and gadolinium-enhanced perfusion MRI,51 may further advance the diagnostic capabilities of MRI. Perfusion MRI may assist in distinguishing between radiographically and symptomatically similar conditions such as bone marrow edema and subchondral insufficiency fractures.52 In pediatric patients with developmental dysplasia of the hip, perfusion MRI was helpful in identifying those at risk for osteonecrosis after closed reduction/spica casting.53 In addition, a whole-body bone scan provides an option for patients at risk for multifocal osteonecrosis, such as those receiving systemic corticosteroids or immunosuppressants.54The early stages of osteonecrosis of the femoral head are frequently asymptomatic but may also present with radiating pain from the hip or groin and limited range of motion of the joint on physical examination.5Bone marrow edema syndrome (BMES) presents as sudden pain without a clear precipitating event. On imaging, BMES shows diffuse edema compared with more localized areas in osteonecrosis. Some studies have suggested that BMES may precede osteonecrosis.5A subchondral insufficiency fracture presents similarly but occurs after an injury. Although both conditions present with low-signal subchondral bands, osteonecrosis imaging presents with a smooth, concave line while the fracture presents with a jagged, discontinuous, convex finding. Conservative treatment is unlikely to improve fracture symptoms, and both conditions can progress to the need for a THA.5Although rare, clear cell chondrosarcoma and chondroblastoma can present with radiolucent lesions in the femoral head. These conditions are not accompanied by the edema present in osteonecrosis or other similar conditions such as BMES.55 The University of Pennsylvania system was developed in an attempt to more clearly delineate the progression of osteonecrosis and to promote distinctions between the stages by adding stage 0 for preradiographic disease, dividing Ficat stage II into two stages based on the absence (II) or presence (III) of a crescent sign, and dividing Ficat IV into two stages: flattening with joint space narrowing only (V) and joint deformity and joint space obliteration (VI).56 The Association Research Circulation Osseous (ARCO) system closely follows Ficat with the exception of the inclusion of MRI findings in stage I and division of stage II based on the extent of femoral head flattening (IIIA if < 2 mm and IIIB if > 2 mm). The ARCO system was recently revised based on an international expert taskforce to better incorporate results of both MRI and plain radiography.57 These staging systems are summarized in Table The most popular staging system for osteonecrosis of the femoral head is the Ficat classification Table . Develop58 For the purposes of patient evaluation and treatment, the most important classification is precollapse versus collapse because this guides discussion of conservative treatment versus THA. For research purposes , we recommend using the updated ARCO guidelines because they effectively use multiple imaging modalities and delineate smaller changes between stages. This allows for a higher level of detail in tracking disease progression and may help to provide clearer answers because the effectiveness of new therapies is evaluated.Systematic analysis of different staging systems found that any classification system is valuable and sufficient for the staging of osteonecrosis, provided necessary data are collected to allow conversion to another metric.59Evaluating risk of progression is important in determining an appropriate treatment choice Table . Althoug60 In combination with observation, restricted weight-bearing is usually advised, although this has not shown utility as a primary treatment modality.61 A study of observation as a strategy in osteonecrosis of the hip has found a failure rate of over 80% by four years and is not recommended as a standalone treatment in advanced lesions.62The most conservative management, observation, has been considered as a possible approach to osteonecrosis. There has been some evidence for spontaneous resolution of small early-stage osteonecrosis lesions.63 Although some early studies showed positive effects of disphosphonates, a recent large multicenter randomized controlled trial found no difference between alendronate and placebo.64 Furthermore, a meta-analysis of five randomized controlled trials had similar findings, with little to no evidence supporting the efficacy of disphosphonates in the nontraumatic osteonecrosis of the femoral head.65 The primary utility of disphosphonates is in the early stages of disease, and they are not preferred to surgery as osteonecrosis progresses.61Medications have been a mainstay of osteonecrosis treatment, but recently, their effectiveness has been questioned. Disphosphonates are a popular choice for pharmacologic treatment and work by inhibiting osteoclast activity. Studies of the use of disphosphonates have shown mixed results.47 increased autophagy,66 suppression of Peroxisome proliferator-activated receptorγ, and activation of the Wnt signaling pathway.67 Statins have been effective in combination with multiple core decompression (CD) procedures, improving both clinical and radiographic progression of osteonecrosis.68Studies have identified multiple potential mechanisms for beneficial effects of statins in delaying osteonecrosis including lipid-lowering effects,63 Hyperbaric oxygen treatments, pulsed electromagnetic fields, and extracorporeal shockwave therapy have been proposed showing some positive outcomes, but disagreement about their effectiveness makes them difficult to recommend.69Several other modalities have been proposed for the treatment of osteonecrosis with varying success. Lipid modifiers such as dietary changes or lipoic acid supplements have shown some positive results in trials, but there is insufficient evidence to recommend them as primary treatment strategies.70 in his early descriptions of osteonecrosis and the CD procedure, noted increased intramedullary pressures, which are released with CD leading to a relief of pain and eventual restoration of blood flow if the lesion is treated early in its progression. Although older studies of CD were equivocal about its effectiveness, study of more recent procedures has shown notable benefits. Studies of both short-term and long-term outcomes have shown improvement in patients treated with CD and delayed time to THA compared with more conservative treatment options.71 As with many treatments, these outcomes are more positive when used in the early stages of disease, with up to 100% of hips surviving 3 years69 and up to 96% surviving 10 years in early-stage disease.71 More precisely, CD has shown positive results in osteonecrosis showing no collapse, a central lesion, and small size (combined necrotic angle <250°).72 These outcomes may prove even more beneficial when paired with grafts and cell-based therapy.CD is done for osteonecrosis of the femoral head to reduce intraosseous pressure and promote increased blood flow and bone genesis. Ficat,73 or a vascularized graft from the iliac crest, fibula, or greater trochanter74 into a core space created for the procedure or from a CD procedure. Nonvascularized bone grafting has shown moderate success, especially with smaller lesions, having a 55% to 87% success rate with a 2- to 9-year follow-up across several studies.69 VBG has shown a 5-year hip survival of 80% in precollapse lesion or 60% after 14 years in similar patients,69 with low conversion to THA.75 However, the benefits of VBG are primarily realized in smaller lesions without notable collapse.76 Ongoing research has evaluated synthetic scaffolds used with or without biofactors to enhance integration and bone growth. Numerous organic, inorganic, and biologic materials have been developed with promise, although no definitive solution has been identified.77Nonvascularized bone grafting involves the placement of bone graft material to provide structural support with the intent of reducing intraosseous pressure and preventing collapse in early stages of osteonecrosis. Vascularized bone grafting (VBG) also seeks to introduce increased blood supply. The graft is done by placing a nonvascularized cortical allograft from the ilium, tibia, or fibula,78 In addition, cell therapy can be combined with other therapies such as CD and/or bone grafts and can potentially improve outcomes.69 A study has shown benefits of bone morphogeneticprotein (BMP) in addition to allograft and/or CD in improving bone formation and limiting the progression on osteonecrosis.79Because osteonecrosis is thought to result from a deficiency of bone regeneration, use of stem cell treatments has been proposed to halt or reverse its pathogenesis. Studies have shown lower rates of radiographic progression and lower need for THA in patients treated with autologous stem cell transplants. In early studies, the combination of autologous stem cell transplant with CD showed a notable delay of an average of 10 years (up to 17 years) in time to collapse.69Osteotomy attempts to delay the progress of osteonecrosis by relieving weight-bearing on necrotic or prenecrotic areas to prevent collapse. To do this, weight-bearing osteonecrotic region is angled or rotated to place primary pressure on a non-necrotic area of the bone. Rotational (82% to 100% from 3 to 15 years) and angular (82% to 98% between 6 and 18 years) osteotomies of the femoral head have shown excellent success rates. However, future THA can become difficult if necessary because of persistent implant and altered anatomy.69Resurfacing of the joints in question is the most minimal option for advanced osteonecrosis and involves replacing the articular surface with artificial materials to preserve natural anatomy. However, because of the complications from materials and possible contribution to osteonecrosis progression, resurfacing is no longer used as osteonecrosis treatment of the femoral head.77 Although patients who have a THA for osteonecrosis have more comorbidities and more complicated hospital stays than those having THA for osteoarthritis, long-term follow-up has shown similar outcome between the two groups for implant survival, osseointegration, and complications such as aseptic loosening.80 Other studies, however, have shown increased rates of sepsis,81 transfusion requirement, and hospital readmission in patients with osteonecrosis who underwent THA compared with OA patients. Recent analysis has shown improved outcomes, with >90% of osteonecrosis THAs surviving 4 to 7 years compared with 8 to 37% survival rates before 1990, possibly because of improved implants and materials used in the procedures.69 The literature is limited in examining etiology-based implant survival, but a study of patients with osteonecrosis secondary to alcohol consumption showed excellent long-term outcomes.82 It is also important to note that the study of patients with osteonecrosis requiring THA found that 46.6% of the patients would go on to require contralateral THA, especially if the contralateral hip had radiographic evidence of osteonecrosis at the time of the first THA, suggesting the need for a close follow-up.83Joint arthroplasty is the only definitive cure for osteonecrosis available at this time; however, potential downsides require careful consideration. THAs are not a permanent solution, and although they may be beneficial early in older patients to reduce cumulative procedures, most patients with osteonecrosis are relatively young. Given this population, if the joint is replaced at diagnosis, the patient will likely need another arthroplasty or revision later in life. Recommendations for joint arthroplasty include advanced disease, continuing progression, and continuing provocative factors.Osteonecrosis continues to be a condition of widely variant etiologies, treatments, and developmental profiles. Because incidence continues to rise, increased understanding of the pathophysiology is necessary to promote developments of new treatments and corrective procedures. Although promising developments are being made in areas such as bone grafting and stem cell therapy, the field continues to lack an agreed-upon regimen to provide patients with osteonecrosis the greatest quality of life and delay their progression to debilitating injury, collapse, or joint arthroplasty. To more effectively understand this disease process, more data are needed. A national registry would be the most complete system to determine diagnostic and treatment directions. In the absence of such a coordinated effort, institutional registries and large cohort studies would help to make advances in this realm.In the area of treatment, there are many potential avenues for improvement. Promising advancements in bone repair such as anabolic agents may play a role in promoting healing. In addition, more directed therapies for coincident conditions may reduce the secondary development of osteonecrosis from steroids and chemotherapy. With an expanded study of etiologies, prevention, and therapy, there is a reason to hope for advancements in reducing the burden of this disease.


Idiopathic pulmonary fibrosis (IPF) is a form of usual interstitial pneumonia (UIP), though its origin is unknown. IPF remains one of the most aggressive and lethal forms of interstitial lung diseases. Extensive basic and clinical research during the past decade has uncovered critical aspects related to the pathophysiological events that lead to the formation of fibrotic scars in the lungs of IPF patients. Our current understanding of IPF pathogenesis includes repetitive/chronic injury to alveolar epithelial type 2 cells (AEC2) that triggers an aberrant wound healing response associated with the activation of fibroblasts and the infiltration of immune cells. This leads to the excessive accumulation of myofibroblasts that deposit extracellular matrix (ECM) proteins, particularly collagen and fibronectin. All aforementioned processes culminate in detrimental scarring of the lung tissue, loss of AEC2 and AEC1, and respiratory failure [bona fide AEC2 (Lysohigh) is the predominant AEC2 subset in the healthy lung, and that this population is largely replaced by an intermediate AEC state (Lysolow) in bleomycin-induced pulmonary fibrosis and in human IPF. Lysolow cells express, in addition to typical AEC2 markers, markers of basal cells, thus highlighting epithelial cell plasticity as a source of cellular heterogeneity in IPF [low cells are also reminiscent of the tdTomato-low (tdTom)low cells, identified using the AEC2 lineage-tracing mouse line  [low cells have been shown to expand during compensatory lung growth following pneumonectomy and were therefore dubbed injury-activated alveolar progenitors (IAAPs) [Fgf10 [In this Special Issue, Wasnick et al. describe two subsets of AEC2 in both healthy and fibrotic lungs [y in IPF ,4,5,6,7.atoflox) ,9,10. Su (IAAPs) ,9. In th (IAAPs) . They al) [Fgf10 . Importa) [Fgf10 . In the ) [Fgf10 . These 3MUC5B promoter rs35705950 polymorphism and the risk of IPF, and the bronchiolization of distal airspaces in IPF lungs. The contribution of airway epithelial cells to IPF pathogenesis is also addressed by Schramm et al., who discuss the role of the ErbB receptor–ligand system in lung fibrosis [Despite the fact that IPF is considered as a “distal” lung disease, with a central role of AEC2 malfunction during its pathogenesis, Chakraborty et al. highlight recent developments that point towards the involvement of airway epithelial cells in IPF pathobiology . The confibrosis . Intrigufibrosis .The cellular heterogeneity of IPF is further discussed by Preisendörfer et al., who stress the potential role of B cells in IPF pathobiology . The patIPF is also associated with a set of metabolic disorders and targeting cellular metabolism, particularly in mesenchymal cells, represents an exciting therapeutic avenue ,19,20,21One of the factors that contribute to the incurable nature of IPF is the fact that patients are already in advanced disease stages at the time of diagnosis. As such, treatment mainly focuses on treating the symptoms and slowing down disease progression rather than treating early events that initiate downstream pathological signaling cascades. Accordingly, efforts that seek to find alternative therapeutic agents against IPF are still ongoing.Current treatment modalities for IPF patients rely on two approved drugs: pirfenidone, which is believed to act as a transforming growth factor beta 1 and hedgehog signaling inhibitor , and ninKorfei et al. contribute to the Special Issue with a comprehensive review on the current knowledge regarding IPF pathophysiology, the associated cellular and molecular mechanisms, clinical management, as well as novel therapeutic targets and therapies in development . ImportaBraubach et al. go beyond IPF and provide a broad characterization of fibroelastotic remodeling (FER), its anatomic distribution, and clinical association . FER is In conclusion, we present a collection of articles in this Special Issue that touch on all aspects of IPF, from the molecular mechanisms driving disease development and progression, to translational models enabling high-throughput drug screening, finishing on clinical studies evaluating adverse effect profiles of drugs approved for IPF treatment. Despite immense improvements in understanding the molecular basis of IPF, further research is urgently needed to pursue innovative translational studies in order to learn more about the enormous repertoire of molecular tricks that lung cells use to adapt to stress conditions and fight against damaging agents.


However, the body of evidence emerging from randomised controlled trials indicates that regional anaesthesia/analgesia has no clinical impact on cancer progression after mastectomies or major abdominal surgery for different cancers.8One of the earliest studies tested the hypothesis that regional anaesthesia could reduce cancer progression by different mechanisms, including modulation of the sympathetic response, avoidance or reduction of general anaesthetics and opioids, and direct immunomodulatory effects of local anaesthetics.9 These effects in vitro and in vivo; however, only 1 retrospective study has evaluated the association between the use of intravenous lidocaine on ovarian cancer progression. Interestingly, the authors reported that the intravenous use of lidocaine was associated with longer survival after ovarian cancer surgery.10 While the results from that study appear to be impactful, there is no evidence from randomised controlled trials supporting the short-term intraoperative use of lidocaine to improve oncological outcomes.It has also been speculated that lidocaine given intravenously during the duration of surgery and postoperatively could also influence cancer progression by enhancing the activity of immune cells such as natural killer cells, reducing the use of opioids, and acting directly on cancer cells in which it would induce apoptotic mechanisms or affect cell motility.11 On the other hand, they suggested that propofol would have the opposite effects. As a result, multiple retrospective studies and meta-analyses were conducted to determine the association between propofol-based intravenous anaesthesia and longer recurrence-free, progression-free, or overall survival after cancer surgery. The results of these investigations are conflicting. For example, Wigmore et al12 indicated that propofol-based intravenous anaesthesia improved the survival of patients undergoing surgery for abdominal metastatic or non-metastatic cancer. Contrarily, Makito et al13 using an extensive Japanese national registry, reported no clinically relevant differences in survival. Unfortunately, no data is available from well-designed randomised controlled trials evaluating the impact of propofol-based intravenous anaesthesia versus volatile-based anaesthesia on cancer recurrence.A group of investigators postulated and proposed the theory that volatile anaesthetics could also have a negative impact on oncological outcomes.14 indicated that propofol had in vitro antiproliferative effects in hepatocellular carcinoma cells. However, the range of doses of propofol (15-120 μM) used in that study was higher than the plasma concentrations of the drug to maintain general anaesthesia (11-22 μM). Therefore, we could conclude that drugs like propofol could have direct anticancer effects in doses associated with adverse effects  in humans. On the other hand, 6-hour exposure to sevoflurane did not affect cell proliferation or metastasis in breast cancer cells.15The question that arises from the current evidence regarding the impact of any anaesthesia technique on cancer recurrence is why there are conflicting results from retrospective studies and less confusing answers from randomised controlled trials. An obvious answer is that retrospective clinical investigations suffer from significant biases, while randomised controlled trials account for most limitations. However, it can also be speculated that a major problem lies in the design of experimental studies investigating the effects of anaesthetics on cancer mechanisms related to tumour growth and metastasis. Such issues include large or repeated dosages of the tested drugs, cultured conditions not resembling the tumour microenvironment, and animals with immunological deficiencies to allow tumour engraftment. For instance, let us examine in vitro and in vivo studies demonstrating the anticancer effects of propofol. Hu et al16 To address this question in the clinical setting, Hovaguimian et al17 conducted a randomised controlled trial in women with breast cancer. They measured natural killer cell activity and circulating tumour cell load before and after surgery under sevoflurane-based general anaesthesia or propofol-based general anaesthesia. The study demonstrated no differences in natural killer cell function or a correlation between function and circulating tumour cell load in both groups of patients.18 More recently, Oh et al19 randomised patients with colorectal cancer to sevoflurane-based general anaesthesia or propofol-based general anaesthesia and measured the fraction and apoptotic rate of circulating immunocytes by flow cytometry. No differences between groups were detected in the fraction of natural killer cells and T lymphocytes.Alternatively, we could theorise that the anticancer effects of intravenous anaesthetics such as propofol could be mediated by a predominant impact on systemic immune effectors against cancer such as natural killer cells, as demonstrated in rodents. In contrast, volatile anaesthetics would impair the function of those cells.20 For instance, a multicentre, double-blind, placebo-controlled, randomised biomarker trial demonstrated that the administration of oral etodolac (400 mg) and propranolol (20 mg) 5 days before the day of the procedure and 5 days after breast cancer surgery reduced systemic inflammation and pro-metastatic biomarkers in the tumour and circulating immunocytes.22 Shaashua et al,23 who included patients undergoing colorectal cancer surgery, used the same strategy in a clinical investigation. Although the study enrolled 34 patients, the authors demonstrated that patients treated with etodolac and propranolol showed a favourable immune profile and a benefit in survival.On the one hand, we could conclude from these studies that short-lived not-targeted perioperative interventions (mainly intraoperative or 1 or 2 days after surgery) do not influence immune makers, cancer cell behaviours, and more importantly, survival as previously implicated. On the other hand, we could hypothesise that pharmacologically targeted interventions administered in the perioperative period for a more extended period (weeks) could influence oncological outcomes. Clinical trials in humans support the evidence for this hypothesis.In conclusion, the use of intraoperative propofol or sevoflurane cannot explain differences in the mechanisms of cancer progression and different rates of survival between patients after cancer surgery. The use of regional anaesthesia should be indicated to provide adequate pain control during and after cancer surgery, but it does not modify patterns of cancer progression or survival. Perhaps, long-term (weeks) perioperative interventions focused on modulating inflammation (anti-inflammatory drugs) and reducing the stress response (beta-blocker) might confer survival benefits for patients undergoing cancer surgery.


EIF2AK4  or in a sporadic form in older age (sPVOD), following exposure to chemotherapy or organic solvents. In contrast to PAH, PVOD is characterized by a particular remodeling of the pulmonary venous system and the obliteration of small pulmonary veins by fibrous intimal thickening and patchy capillary proliferation. The pathobiological knowledge of PVOD is poor, explaining the absence of medical therapy for PVOD. Lung transplantation remains the only therapy for eligible PVOD patients. As we recently demonstrated, respiratory diseases, chronic obstructive pulmonary disease, or cystic fibrosis exhibit lipointoxication signatures characterized by excessive levels of saturated phospholipids contributing to the pathological features of these diseases, including endoplasmic reticulum stress, pro-inflammatory cytokines production, and bronchoconstriction. In this study, we investigated and compared the clinical data and lung lipid signature of control (10 patients), idiopathic PAH (7 patients), heritable PAH (9 BMPR2 mutations carriers), hPVOD (10 EIF2AK4 mutation carriers), and sPVOD (6 non-carriers) subjects. Mass spectrometry analyses demonstrated lung lipointoxication only in hPVOD patients, characterized by an increased abundance of saturated phosphatidylcholine (PC) at the expense of the polyunsaturated species in the lungs of hPVOD patients. The present data suggest that lipointoxication could be a potential player in the etiology of PVOD.Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary arterial hypertension (PAH) occurring in a heritable form (hPVOD) due to biallelic inactivating mutations of  Cell membranes are mainly constituted of phospholipids (PLs) as combinations of several classes and molecular species, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), or lysophosphatidylcholine (LPC), which differ as a function of their polar head group, but also according to the length and degree of unsaturation of their acyl chains . PLs acyBMPR2), induced by drugs or toxins, or associated with overt features of venous/capillaries . PVOD represents a rare form of PAH characterized by the preferential involvement of the pulmonary venous system and the obliteration of small pulmonary veins by fibrous intimal thickening and patchy capillary proliferation. PVOD is due to biallelic mutations in EIF2AK4  encoding for general control nonderepressible 2 (GCN2). A sporadic form of PVOD (sPVOD) is described mainly in the context of exposure to specific chemotherapeutic agents or organic solvents  pati pati2  and FASN (fatty acid synthase) [−/−gcn2 mice exposed to high fat diet, Liu et al. demonstrated an increase in the expression of genes involved in lipolysis and a reduced expression of fatty acid gene synthesis in the liver from gcn2−/− mice [Furthermore, GCN2 regulates fatty-acid homeostasis, since e liver. . gcn2−/−ynthase) . Moreove−/− mice . These rWhatever the origin of these alterations, it is likely that this lipointoxication will not be without any consequences on lung function in hPVOD patients. Indeed, we have already demonstrated that similar alterations dramatically affect crucial cellular processes, both in bronchial epithelial cells  and The number of samples used in this study is relatively low (9–10 patients), but PVOD is a subgroup of a rare disease, so it was difficult to obtain more samples from these subgroups.2+-independent groups.Concerning the hypothetical involvement of PLA2 in lipointoxication observed in hPVOD patients, further studies are needed to quantify the level of expression and the activity of PLA2 to determine the involvement of PLA2 isoforms in hPVOD pathogenesis, since the PLA2 superfamily is described to be composed of five families defined by their physiological roles, including secretory PLA2, cytosolic PLA2, lysosomal PLA2, and two major PLA2 CaMoreover, in this analysis, our cohort is predominantly male, while PAH or hPVOD are generally characterized by female predominance, contrary to the results for sPVOD. As we compared sPVOD to other forms , we selected similar male predominance, including in control patients, to be comparable. Finally, our analyses did not find any differences between males and females.To summarize, for future studies to determine the role of GCN2 deficiency in lung lipointoxication, the present data suggest that lipointoxication could be a potential player in the etiology of hPVOD, but further additional studies are needed to understand the mechanisms explaining lipointoxication in hPVOD and to identify the significance of lipointoxication in its development.


The primary cilium is a hair-like, microtubule-based organelle that is covered by the cell membrane and extends from the surface of most vertebrate cells. It detects and translates extracellular signals to direct various cellular signaling pathways to maintain homeostasis. It is mainly distributed in the proximal and distal tubules and collecting ducts in the kidney. Specific signaling transduction proteins localize to primary cilia. Defects in cilia structure and function lead to a class of diseases termed ciliopathies. The proper functioning of primary cilia is essential to kidney organogenesis and the maintenance of epithelial cell differentiation and proliferation. Persistent cilia dysfunction has a role in the early stages and progression of renal diseases, such as cystogenesis and acute tubular necrosis (ATN). In this review, we focus on the central role of cilia in kidney development and illustrate how defects in cilia are associated with renal disease progression. Cilium emanates from the mother centriole, and it is classified into motile cilia (9 + 2 structure) and nonmotile cilia (9 + 0 structure). Motile cilia have nine peripheral doublets of microtubules with a central pair complex (9 + 2), and primary cilia lack the centrally located pair (9 + 0) , 2. In vThe cilia life cycle is tightly related to the cell cycle –11 and cCompared to cilium assembly, the signaling pathways for cilia disassembly are poorly understood . The AurCiliary membranes are rich in receptors and ion channels that can be activated by mechanical or chemical stimuli . The pro-/-Wnt9b and -/-Wnt4 mice. However, it is important to maintain a proper balance of canonical Wnt signaling activity, and constitutive β-catenin activation results in cyst formation in all tubular segments  as it invades and branches into the metanephric mesenchyme (MM) and acts as a paracrine signal to induce the expression of tubulogenic pathway markers, such as fibroblast growth factor-8 (FGF8), Wnt4 and Pax8 .. Wnt9b segments . Ksp-creithelium . Module ithelium .via β-catenin-dependent pathways in the absence of flow  polPKD1 and PKD2 (PKD1 and PKD2 encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Colocalization of GPCR PC1 and ion channel PC2 mediates flow-sensitive mechanotransduction in primary cilia and responds to flow by increasing calcium influx .ARPKD occurs mainly in infants and young children, with a prevalence of 1:20,000 . It is cNPHP1, NPHP2/inversin, NPHP3, NPHP4, NPHP5, NPHP6/CEP290, NPHP7/GLS2, NPHP8/RPGRIP1L, NPHP9/NEK8) have been implicated in NPHP. Unidentified mutated genes still need to be explored in 70% of cases (Nephronophthisis (NPHP) is an autosomal recessive disease that accounts for 10%-20% of cases of renal failure in children. It is characterized by cystic kidney tubules and interstitial fibrosis with inflammatory infiltrate. NPHP-related genes  mutant mice have shorter and blunted primary cilia in collecting ducts  is a clinical syndrome of rapid decline in kidney function over a short period of time (a few hours or days), resulting in the retention of metabolic waste products, urea and creatinine , 62. AcuElizabeth Verghese et al. identified that acute tubular necrosis causes an increase in the length of renal cilia, modifying their sensory sensitivity during repair . BiopsieTTC21B causes familial primary FSGS, and knockdown of the TTC21B gene product IFT139 in podocytes leads to primary cilia defects and abnormal cell migration  is a pathologic diagnostic term that is mainly characterized by sclerosis of part of the glomerular  or part of the glomerular capillary loops  . Evelyneigration . NeverthIvana Solic et al. recently summarized the length of the primary cilium between healthy control and pathologically-changed kidney tissues, including FSGS, CSF and MCDK. In MCDK, CNF and FSGS, cilia were significantly elongated compared to healthy controls .There are still many cilia-related phenotypes and functions that need to be explored in the near future in IgA nephropathy and membranous nephropathy.Lupus nephritis represents the most severe clinical manifestation of systemic lupus erythematosus (SLE) and leads to a high percentage of morbidity and mortality in patients. The etiology of SLE is characterized by interactions between genetic susceptibility, immune system abnormalities and hormone regulation disorders that result in tolerance disorders and sustained autoantibody production . Numerouvia “antennas” on the surface of RTECs and primary cilia, which direct the metabolic reprogramming of cells to adapt to the environment (Diabetic kidney disease (DKD) is one of the most serious complications of diabetes and has become the most common cause of ESRD worldwide . Studiesironment . HoweverThe primary cilium of RTECs is an important mechanical force sensor for the shear stress, regulates the energy metabolism homeostasis in RTECs to ensure the energy supply for reabsorption function . DiabetiIn patients with RCC with mutations in the VHL (von Hippel–Lindau disease tumor suppressor) gene, primary cilia have been lost, and the re-expression of VHL proteins restored cilia occurrence . Cilioge2+. The CT-Fe2O3-NPs significantly improved cardiac function in the ciliopathic hypertensive models , a cytoplasmic enzyme, is the major driver of cilium disassembly, and small molecules that inhibit HDAC6 have been demonstrated to restore the ciliary defective phenotype –90. AntiIn summary, the primary cilium is the center platform that regulates diverse developmental signaling pathways, and its function relies on the control of the precise dynamic balance between cilia assembly and disassembly. More details of these signaling pathways and their involvement in kidney disease remain to be explored. How cilia sense mechanical stimuli is still an ongoing research topic. Ciliary-targeted technology urgently needs to be developed. Insights into ciliary defects in kidney disease will help us identify therapeutic targets for kidney injury relief and provide novel insights into disease mechanisms and ciliopathy intervention.YB conceptualized and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (Grant No. 32141005 and 82070741).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.


Tumor suppressor genes (TSGs) are often involved in maintaining homeostasis. Loss of tumor suppressor functions causes cellular plasticity that drives numerous types of cancer, including small-cell lung cancer (SCLC), an aggressive type of lung cancer. SCLC is largely driven by numerous loss-of-function mutations in TSGs, often in those encoding chromatin modifiers. These mutations present a therapeutic challenge because they are not directly actionable. Alternatively, understanding the resulting molecular changes may provide insight into tumor intervention strategies. We hypothesize that despite the heterogeneous genomic landscape in SCLC, the impacts of mutations in patient tumors are related to a few important pathways causing malignancy. Specifically, alterations in chromatin modifiers result in transcriptional dysregulation, driving mutant cells toward a highly plastic state that renders them immune evasive and highly metastatic. This review will highlight studies in which imbalance of chromatin modifiers with opposing functions led to loss of immune recognition markers, effectively masking tumor cells from the immune system. This review also discusses the role of chromatin modifiers in maintaining neuroendocrine characteristics and the role of aberrant transcriptional control in promoting epithelial-to-mesenchymal transition during tumor development and progression. While these pathways are thought to be disparate, we highlight that the pathways often share molecular drivers and mediators. Understanding the relationships among frequently altered chromatin modifiers will provide valuable insights into the molecular mechanisms of SCLC development and progression and therefore may reveal preventive and therapeutic vulnerabilities of SCLC and other cancers with similar mutations. Investigating how mutated protein complexes trigger immune evasion during small-cell lung cancer (SCLC) could yield possible therapeutic approaches for this intractable disease. SCLC is driven by loss-of-function mutations in tumour suppressor genes, which in turn alter the activity of chromatin modifiers, protein complexes that alter chromatin, the mixture of DNA and proteins forming chromosomes. Kwon-Sik Park at the University of Virginia in Charlottesville, USA, and co-workers have reviewed current understanding of the mechanisms involved. Studies show that imbalances in different chromatin modifier groups can result in the loss of tumour recognition markers, effectively hiding tumour cells from the immune system. The hyperactivity of one specific chromatin modifier regulates cancer cell plasticity, shifting cells from epithelial to a less specialised type, meaning they can differentiate and spread through the body more quickly. The high mortality of SCLC is mainly due to its highly proliferative and metastatic nature and its rapid development of resistance to genotoxic chemo/radiation therapy. Unlike lung adenocarcinoma, which is frequently driven by druggable alterations such as activating mutations in EGFR, ALK, and KRAS7, SCLC is driven mostly by unactionable, loss-of-function mutations in genes encoding tumor suppressors, including RB1 and P538.Lung cancer is the leading cause of cancer-related deaths worldwide. The two main types of lung cancer are small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC); the latter is further divided into lung adenocarcinoma, squamous-cell carcinoma, and large-cell carcinoma. These lung cancer types are typically defined by unique immunohistological features and are clinically managed very differently. SCLC accounts for 10 to 15% of all lung cancers9. The most striking feature of the SCLC genome is the fact that both the TP53 and RB1 genes are mutated in over 90% of patients9. Studies of genetically engineered mouse models have shown that concomitant loss of Rb1 and Trp53 is required for SCLC initiation but have also suggested roles of additional drivers in progression to full malignancy. A group of chromatin modifiers mutated in 50% of SCLC patient tumors have emerged as putative tumor suppressors given the global impact of their inactivation and their implication in other cancer types10. They include lysine-specific histone acetyltransferases , lysine-specific histone methyltransferases , lysine-specific histone demethylases  and components of the SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex . The small numbers of patient tumors examined in the studies of the SCLC genome preclude a robust analysis of mutual exclusivity and cooccurrence among the mutations to determine functional relationships; however, the known functional interactions among the proteins on target chromatin led to speculation that the epigenetic dysregulation resulting from the mutations is related to key pathways causing malignancy.Recent sequencing studies of the SCLC genome have uncovered a plethora of mutations that may hold the keys to molecular changes within patient tumor cells13 and studies of preneoplasia progressing to full-blown SCLC. More importantly, much less is known about the impact of mutations in mutant epithelial cells on the stroma of patient tumors. SCLC patients rarely undergo surgical resection of primary tumors because of the high occurrence of metastasis16. The limited access to patient tumors makes it difficult to gain direct insights into the SCLC tumor microenvironment (TME). To reveal interactions within the TME, synthesis of data from patient tumors and various mouse models is needed to recapitulate the interactions between tumor cells and cellular components of the microenvironment.Little is known of the specific dysregulated pathways, except for findings from studies of cell cycle dysregulation in cancer cells-of-originHere, we review recent studies suggesting that the balanced actions among various chromatin modifiers are a critical gatekeeper of SCLC cell identity and homeostatic interactions with TME. Alterations in chromatin modifiers largely cause epigenetic dysregulation, leading to transcriptional silencing of immune markers on the surface of mutant cells and effectively leading to immune exclusion within the TME. Concurrently, transcriptional reprogramming enhances cellular plasticity, contributing to the transition between SCLC subtypes and the acquisition of mesenchymal characteristics.17. ICB employs antibodies to block immune checkpoint ligands  to activate cytotoxic T cells (CD8-positive), which induce lysis and killing of target cells19. Theoretically, SCLC seems to be a perfect candidate for ICB: the high mutational burden of SCLC should present sufficient neoantigens for activating the adaptive immune system, rendering these cells susceptible to ICB-driven immunotherapy20. Disappointingly, however, ICB treatment is often ineffective in treating SCLC22. This is largely due to the “immune cold” nature of SCLC tumors, which are characterized by significantly decreased immune cell infiltration. In an early study assessing levels of immune infiltration within patient samples, low-grade tumors showed higher levels of infiltrating CD8+T cells and macrophages than their high-grade counterparts23. Tumors derived from a mouse model of SCLC exhibited alarmingly low levels of immune infiltration, whereas KRAS- and EGFR-driven NSCLC tended to have robust levels of immune infiltration24. Furthermore, the immune cells that do penetrate SCLC tumors tend to contribute to an immunosuppressive environment; for example, there are increased proportions of regulatory T cells (Tregs), M2-polarized macrophages, and myeloid-derived suppressor cells (MDSCs)25. These cells suppress the activation of other immune effectors by releasing anti-inflammatory cytokines, allowing for the unchecked proliferation of tumor cells. The molecular changes within SCLC cells that alter the immune microenvironment have been of significant interest yet remain poorly understood. Particularly, a role of epigenetic alterations in reshaping the immune microenvironment has recently begun to emerge.Recently, interest has arisen around the potential use of utilizing one’s own immune system to target SCLC through immunotherapies, including immune checkpoint blockade (ICB)28. Recent studies have implicated polycomb repressive complex 2 (PRC2) in suppressing MHC-I expression and presentation. Deletion of either embryonic ectoderm development (EED) or enhancer of Zeste homolog 2 (EZH2), the core components of PRC2, was sufficient to restore MHC-I expression and cell surface display29. EED is involved in recruitment of the other subunits to form PRC2, and EZH2 is a catalytic subunit with histone methyltransferase functions. EZH2-mediated trimethylation of lysine 27 on histone H3 (H3K27me3) in gene promoters and enhancers stimulates DNA compaction. This modification of the histone results in a condensed chromatin structure and thus renders the nearby genes inaccessible to transcriptional machinery, leading to gene repression or silencing30. These transcriptional alterations result in insufficient MHC-I display on the cell surface. Furthermore, this activity is often co-opted by many cancers with silencing of various tumor suppressor genes, among which are the genes encoding cell cycle regulators and antigen processing and presentation pathway components. PRC2 was also found to silence genes encoding components of the antigen presentation pathway (APP), including TAP1/2, which are responsible for antigenic peptide transportation to the endoplasmic reticulum (ER) and loading onto assembled MHC-I32. Without proper antigen presentation, tumor-associated antigens can no longer be recognized by and subsequently activate T cells. Epigenetic suppression of MHC-I and APP components prevents SCLC cells from being recognized by T cells, particularly cytotoxic T cells, which are the key effectors of immune checkpoint blockade (ICB) therapy. However, it is important to note that losing the expression of this “self-recognition” receptor should render cells susceptible to being targeted by the cellular effectors of the innate immune system, including macrophages, natural killer (NK) cells, and neutrophils. How tumor cells escape this new challenge remains poorly understood.An increasing body of evidence suggests that chromatin modifiers influence how SCLC cells interact with both the adaptive and innate immune systems. Although ubiquitously expressed, the expression of MHC-I (major histocompatibility complex-I), the primary molecule for engaging cytotoxic T cells, is mostly decreased in SCLC tumors34. Since such polarization results in loss of the antitumor activity of macrophages, the mechanisms underlying the depletion or inactivation of macrophages remain an important research topic. A recent study suggested a role for CD47 on SCLC cells in evasion of macrophages33. CD47 is a cell surface protein on tumor cells that binds to SIRPα  on macrophages, and the protein-protein interaction activates the cytoplasmic domain of SIRPα to trigger intracellular signals leading to inhibition of phagocytic activity36. Elevated levels of CD47 provide a clear example of transcriptional dysregulation in SCLC contributing to immune evasion, but the mechanisms underlying upregulation of this immune marker in SCLC remain unknown. Beyond the expression of CD47, macrophage activation and chemotaxis to tumors is also mediated by chemokines and cytokines37. SCLC tumors with low levels of macrophage infiltration have been found to have low levels of CC chemokine ligand 2 (CCL2) expression, which is a chemokine responsible for the recruitment of monocytes that differentiate into macrophages. Similar to MHC-I, CCL2 was found to be epigenetically silenced by histone methylation at its enhancer mediated by EZH238. In the same study, CCL2 expression was also repressed by DNMT1 methylation of CpG islands at the CCL2 enhancer. Implementation of EZH2 or DNMT1 inhibitors was found to rescue CCL2 expression in SCLC cells. Furthermore, the use of these inhibitors led to increased macrophage recruitment in vivo implantation experiments.Macrophages were found to be largely absent in the immune microenvironment of higher grade SCLC tumors, and those that were present were positive for CD163, indicative of an M2 polarized state of cells39. Upon recognition and activation, NK cells release perforin and granzymes to induce cytolysis of target cells. Compared to NSCLC cells, SCLC cells have significantly lower levels of NKG2DL40. The levels of NKG2DL are negatively correlated with the levels of MYC, a marker of the NEUROD1 subtype of SCLC. MYC works in concert with histone deacetylase 3 (HDAC3) to deacetylate the promoter of NKG2DL  at histone 3 lysine 9 (H3K9Ac), thus removing the transcriptional activation signal. NKG2DL is also recognized by cytotoxic T cells, providing yet another example of epigenetic silencing within SCLC cells that hides them from various components of the immune system. The relationship between NKG2DL and MYC is an example of how the heterogeneity underlying each SCLC subtype contributes to varying levels of immune infiltration within patient tumors.NK cells are another potent effector of the innate immune system. They recognize natural killer group 2, member D ligand (NKG2DL) (including the subsets MICA/B and ULBP) on host cells41, allowing the excessive and sustained proliferation of mutant cells, and is often acquired through aberrant transcriptional regulation of key immune recognition markers  characteristics in SCLC. Furthermore, the use of the pan-HDAC inhibitor trichostatin A (TSA) was also sufficient to increase NK-cell recruitment and activation in allograft experiments43. Together, these studies highlight that the use of inhibitors of chromatin modifiers not only changes the expression of several key immune regulatory markers on the surface of SCLC tumor cells but also leads to increased levels of immune infiltration. The combination of an EZH2 inhibitor with a PD-L1 blocker was suggested after examination of an SCLC cohort in which most patients displayed a stark lack of PD-L1 expression while harboring higher levels of EZH244. When combined with existing ICB strategies, targeting these epigenetic regulators may prime the cells for increased recognition by both the adaptive and innate arms of the immune system. While these aberrant epigenetic modifications in SCLC may present interesting targets for therapeutic intervention, it is important to recognize some of the major caveats of epigenetic therapy. These include a lack of specificity both in delivery and in targeting. An early study evaluating the effects of 5-aza-CdR, an inhibitor of DNA methylation, and TSA, an inhibitor of histone deacetylation, laid out an important framework for testing the effects of epigenetic targeted drugs: comparing the results of parallel genetic manipulation of targeted chromatin modifiers to chemically induced outcomes. In the study, the researchers compared the effects of these nonspecific inhibitors with the effects of targeted knockdown of DNMT1 and DNMT345. The study showed the importance of using genetic targeting strategies, such as CRISPR technology, as a means to assess the impact of directly inhibiting a target gene versus the off-target effects of a nonspecific inhibitor.The significant impact of dysregulated chromatin modifiers on immune evasion and depletion in the TME led to the concept that reversing these transcriptional reprogramming events may be a valuable treatment strategy to be employed in combination with existing ICB. EZH2 has been shown to induce widespread changes in the expression of immune markers in SCLC cells, for example, silencing MHC-I and CCL2, thus inhibiting interactions with T cells and macrophages. Use of an EZH2 inhibitor led to a reversal of MHC-I silencing in an SCLC cell line46. Alternatively, these subtypes can be further subdivided into neuroendocrine (SCLC-A/-N) and nonneuroendocrine (SCLC-P) subtypes. SCLC arises from neuroendocrine cells in the central airway12. The neuroendocrine characteristics are often lost during SCLC progression  to non-NE characteristics). Previously, a fourth subtype, SCLC-Y, was proposed based on YAP1 expression, but the relevance of this subtype has since been questioned, and SCLC-Y is currently regarded as a non-NE subtype rather than an independent subtype. Instead, another subtype called the “inflamed” subtype (SCLC-I) has been added to the list, which is characterized by a gene signature with elevated levels of immune checkpoint markers coinciding with increased levels of immune infiltration47. Levels of immune infiltration differ by subtype, which will be described in further detail below. Recent emphasis has been placed on discovering what drives the progression of each subtype and the overlapping transformation mechanisms that all subtypes use. Epigenetic control of gene expression culminates in alterations made to the chromatin landscape impacting gene expression. The following sections will describe how aberrant chromatin modifiers drive changes to the chromatin layout within transforming SCLC cells to promote tumor development.SCLC has long been treated as a singular disease. This is reflected in the near universal use of the combination of cytotoxic chemotherapy and radiation to treat SCLC patients. This strategy has proven to be mostly ineffective at halting tumor progression. However, there has been a significant shift in the field with the increasing appreciation of the heterogeneity between and within patient tumors. A molecular subtyping strategy that classifies SCLC cells into three groups  based on the expression of neural lineage-defining transcription factors  has recently been proposed50. Methylation of H3K4 is a well-established marker of actively transcribed genes54 Removal of these posttranslational modifications inhibits recruitment and interactions with chromatin remodelers and transcription factors. LSD1 expression was found to be higher in the majority of SCLC samples (at an astounding rate of 98%) compared to adjacent normal tissues, and the protein activity was found to be concentrated at neuronal differentiation genes55. Treatment of SCLC cells with an LSD1 inhibitor revealed that sensitivity to this drug could be predicted by the expression levels of members of the MYC family of transcription factors, including MYCL, MYCN, and MYC. Consistent with the patterns of MYCL/MYCN amplification in the SCLC-A subtype and MYC amplification in the SCLC-N subtype46, the expression of ASCL1 and NEUROD1 in SCLC cell lines representing the SCLC-A and SCLC-N subtypes was dependent on LSD1 activity56. Furthermore, LSD1 inhibition impacted the expression of SYP and CHGA, additional NE markers. Blocking LSD1 activity not only decreased the expression of NE markers but also subsequently resulted in activation of the NOTCH signaling pathway, which indicates transcriptional reprogramming toward a non-NE phenotype50.One of the factors deciding SCLC subtype is lysine demethylase 1 (LSD1), which acts as a transcriptional repressor by removing activating monomethylation or dimethylation of histone H3 lysine 4 (H3K4me1/me2) at regions flanking transcription start sites46. ASCL1 expression is often sustained by chromatin modifiers. Similar to LSD1, when the histone demethylase KDM5A was targeted by CRISPR/Cas9 in various SCLC cell lines, the expression of ASCL1 and the NE markers CHGA and SYP decreased57. These SCLC cell lines often grow as clusters in suspension; however, after targeting KDM5A, the cells became adherent in culture. This transition in culture is often a sign of SCLC cells losing their NE characteristics. Furthermore, KDM5A was found to bind to the NOTCH2 promoter and to several NOTCH target genes, silencing their expression and silencing the overall NOTCH signaling pathway. LSD1 and KDM5A are examples of proteins that epigenetically control cellular identity by sustaining the NE features of SCLC cells.ASCL1 is a key transcription factor in the maintenance of NE features of SCLC cells and is highly expressed in 70% of SCLC cases58. NE-high tumors (SCLC-A/-N) were found to have decreased levels of T-cell and NK-cell infiltration compared to NE-low tumors (SCLC-P). These low levels of immune infiltration were in contrast with the increased levels of T cells, NK cells, and macrophages within NE-low tumors. These tumors represent the emerging inflamed subtype (SCLC-I)47. Genetically engineered mouse models (GEMMs) of the two NE-high subtypes of SCLC 13 recapitulate what is seen in patient samples. The tumors from the RP and RPR2 GEMMs, both of which are SCLC-A, displayed higher levels of immune markers than the RPM GEMM  tumors, which are SCLC-N. The levels of immune infiltration within these tumors also followed the patterns seen in patient tumors, with RPM tumors displaying the lowest amount of T-cell infiltration58. The analysis of patient data and validation in GEMMs confirms the classification of SCLC as being immune cold, with the NEUROD1 subset of tumors showing the greatest lack of infiltrating immune cells.The previous section described the relative lack of immune cells within the SCLC microenvironment. Further examination of the correlation between immune infiltration within patient tumors and circulating levels of immune cells revealed that immune cell infiltration within the tumor varies based on the degree of NE characteristics of the tumor25, contributing to an immunosuppressive environment. These results highlight the heterogeneity of immune infiltration composition based on SCLC subtype: NE-high subtypes (SCLC-A/SCLC-N) are primarily immune-suppressive based on immune cell infiltration levels and surface levels of immune recognition markers. While these NE characteristics have been shown to be sustained through the efforts of various chromatin modifiers, including LSD1 and KDM5A, how epigenetic regulation of NE features contributes to the varying expression levels of immune markers remains to be determined.Not only did NEUROD1 tumors have the lowest levels of infiltrating immune cells, but the T cells present were also found to be composed of higher levels of regulatory T cells (Tregs) and cytotoxic T cells (CD8+) displaying an exhausted phenotype than the respective levels in SCLC-A tumors29. Interestingly, SCLC patient samples with low levels of NE markers  tended to have high levels of MHC-I expression that correlated with the expression of EMT markers59. In the same study, the researchers discovered a subset of cells in the H69 SCLC cell line that were enriched for low NE expression and an increase in TAP1 and MHC-I expression. Rescue of the expression of these markers was correlated with an increase in levels of H3K27Ac, a transcriptional activation marker opposing the repressive markers of PRC2. This suggests that as SCLC cells transition from an NE to a non-NE phenotype, they undergo transcriptional reprogramming, which impacts not only lineage plasticity but also immunogenicity. This notion was further exemplified by a recent study assessing the effects of LSD1 inhibition in various SCLC cell lines60. LSD1 inhibition led to a loss of NE features that coincided with the rescue of the expression of MHC-I and other components of the APP pathway. LSD1-mediated restoration of MHC-I and APP component expression sensitized RPR2 tumors to PD-L1 blockade, resulting in increased infiltration of cytotoxic T cells. This study provides a clear example of the efficacy of targeting chromatin modifiers to treat SCLC by enhancing the response to ICB. It also provides a novel link between the simultaneous regulation of NE characteristics and immune recognition markers in SCLC.NE-high tumors consistently show high levels of immune exclusion due to low expression levels of MHC-I and TAP1, both of which are epigenetically silenced by PRC262. This alarmingly high rate of metastasis presents a therapeutic challenge. It has been suggested that the metastatic switch in SCLC is driven by “chromatin reprogramming”63. This idea was supported by an analysis of patient samples and tumors derived from RP GEMMs in which the expression of the neural transcription factor NFIB was increased in metastases66. Although the mechanism remains undiscovered, the elevated levels of NFIB correlated with a hyperaccessible chromatin state. Interestingly, in melanoma, NFIB was found to increase EZH2 expression67. As described in the previous section, EZH2 is a potent regulator of epigenetic dynamics in SCLC. Chromatin modifiers control transcription by fostering either an open or closed conformation. Closed chromatin, often resulting from amplified levels of histone methylation, is marked by an increase in DNA compaction around histones and thus renders these regions of the genome inaccessible to transcriptional machinery. In contrast, acetylation of histones along with nucleosome sliding or even eviction increases accessibility. Anomalous control of these accessibility states is a key mechanism by which chromatin modifiers drive cellular plasticity.At the time of diagnosis, two-thirds of SCLC tumors have already metastasized69. However, the mechanism behind these selective methylation patterns remains unknown. In the previous section, this pattern was shown to manifest in the loss of cellular identity, including the loss of NE characteristics and immune markers. This portion of the review will discuss how imbalance of chromatin modifiers propels the transformation from an epithelial to a mesenchymal state. Epithelial-mesenchymal transition (EMT) is a major cellular mechanism underlying metastasis and is driven by a number of genetic and phenotypic changes. These changes are mainly related to cell-cell interactions resulting in increased mobility, allowing tumor cells to migrate and invade secondary regions throughout the body. The most common EMT- associated changes include loss of expression of the cell adhesion molecule E-cadherin (CDH1) and upregulation of the expression of N-cadherin (CDH2) and vimentin70. Furthermore, these transcriptional changes are maintained through increased expression of the following EMT-associated transcription factors: SNAIL, SLUG, TWIST, ZEB1, and ZEB270. Just as the transition between SCLC subtypes is being increasingly appreciated as a gradual process in which transitioning cells often display overlap of subtype markers, EMT is no longer regarded as a binary event and has begun to be defined as a progressive transformation70. In accordance with the guidelines from ‘the EMT International Association’ (TEMTIA), the ability of epithelial cells to undergo this transformation will be referred to as “epithelial-mesenchymal plasticity” (EMP) for the remainder of this review.There appears to be a pattern emerging: the shifts in chromatin organization during tumor progression impact the transcription of key genes; regions of the genome containing the promoters of crucial oncogenes tend to be maintained in a hypomethylated (accessible) state, while regions that include the promoters of tumor suppressor genes tend to be hypermethylated (inaccessible)71. This pattern of early metastases is recapitulated in experimental models of SCLC, including GEMMs, which often feature very few initiating mutations: the RP model has only conditional loss of Rb1 and Trp53, the RPR2 model has additional deletion of Rbl2 , and the RPM model additionally features a hyperactive form of MYC13. In particular, cells isolated from the primary tumors of these mice initially grow as spheroid aggregates in suspension, thus indicating that these primary tumor cells alter cell-matrix and cell-cell interactions and display a cellular phenotype conducive to invasion. While the paucity of mutations may support the notion of an inherent capacity of mouse SCLC to metastasize, the nearly intact cell-matrix and cell-cell interactions seen in Rb1/Trp53-deficient preneoplastic cells , derived from early-stage lesions in GEMMs, imply the need for additional drivers to induce drastic cellular transformation73. A recent review laid out the mechanisms of metastasis in SCLC74; however, this section will focus on how epigenetic dysregulation, particularly that induced through the actions of chromatin modifiers, drives EMP in SCLC through epigenetic silencing of epithelial markers and a subsequent increase in mesenchymal characteristics.While metastasis is often associated with the later stages of cancer progression, the tendency for SCLC to exhibit early metastasis has raised the question of whether these cells contain an inherent propensity to metastasize8. Prolonged CSE of human bronchial epithelial cells (HBECs) has also been found to induce aberrant transcriptional control, promoting the transition to a mesenchymal phenotype77. These smoke-exposed HBECs acquire a mesenchymal phenotype, as indicated by elongation. At the molecular level, these cells lack CDH1 expression while simultaneously gaining expression of vimentin and CDH2, both of which are mesenchymal markers. The expression levels of several lncRNAs, including HOTAIR and MALAT1, are increased in HBECs after smoke exposure78. Just as chromatin-modifying complexes control transcription by altering DNA-histone interactions, lncRNAs can regulate transcription through recruitment and assembly of various transcription factors and chromatin-modifying complexes79. Further exploration of the effects of continuous smoke exposure on HBECs revealed that the transcriptional reprogramming was driven by increased EZH2 activity mediated by the lncRNA metastasis-associated in lung adenocarcinoma transcript 1 (MALAT1)80. CSE was found to be positively correlated with MALAT1 expression, which was found to be necessary for maintaining EZH2 expression. Previous studies have provided a link between prolonged smoke exposure and acquiring a mesenchymal phenotype. Furthermore, these studies showed a correlative relationship between CSE and increased EZH2 expression and raise an interesting question: can chronic CSE-induced epigenetic dysregulation prime untransformed NE cells for EMT occurring after the acquisition of additional oncogenic alterations?SCLC patients often have a history of smoking. Chronic cigarette smoke exposure (CSE) has been linked to inducing mutations involving C:G > A:T transversions7. However, in SCLC, the alterations tend to involve the gradual accumulation of loss-of-function mutations. EZH2 hyperactivity has been proven to have widespread effects on transcriptional reprogramming in SCLC. Its apparent hyperactivity may be attributed to frequent loss-of-function mutations in the chromatin-modifying complexes that antagonize EZH2 and compete for occupancy at regulatory regions of the genome. Chromatin modifiers form interconnected, complex networks that often work in concert with or in opposition to each other. PRC2 and methyltransferases are often associated with gene silencing, particularly through methylation of lysine residues 9 and 27 on histone 3 (H3K9/27). In contrast, histone acetyltransferases, such as CREBBP/EP300 , work together with various complexes, such as the SWI/SNF complex, to promote active transcription by relaxing DNA around histones and increasing accessibility to transcriptional machinery. CREBBP was found to be a potent tumor suppressor in SCLC73. Loss of CREBBP-mediated histone acetylation results in transcriptional reprogramming within Rb1/Trp53-deficient preSCs and promotes tumorigenic transformation of these preneoplastic cells. The actions of CREBBP and PRC2 often converge at the regulatory regions of the same genes. EZH2 silences CDH1 expression by methylating histones at its promoter (resulting in H3K27me3)81. In preSCs, targeting CREBBP is sufficient to reduce the expression of CDH1 and tight junction proteins and concomitantly increase the expression of mesenchymal markers, including vimentin and ZEB1. The decrease in CDH1 expression caused by the loss of CREBBP parallels the reduction in CDH1 caused by EZH2-mediated promoter methylation. These findings support the idea that EZH2 hyperactivity in SCLC is crucial to the loss of competitors for chromatin occupancy. Given the high frequency of chromatin modifiers harboring mutations in SCLC tumors, there is a need to investigate how interactions between these complexes drive tumor development and progression through transcriptional reprogramming.In many cancers, such as NSCLC, oncogenic alterations involve the upregulation of potent drivers, such as KRAS and EGFR82, thus providing a potential vulnerability that can be targeted therapeutically. Increased expression of both CDH1 and SLFN11, a helicase involved in the response to DNA damage, was found to be a reliable predictor of sensitivity to PARP inhibitors, suggesting a negative association between EMT and drug sensitivity83. Although that study provided evidence of actionable predictors of therapeutic sensitivity in SCLC, it also reflects a commonality in treating SCLC, in which patient tumors show an initial yet fleeting response to treatment. SLFN11 expression was found to be frequently repressed in cell lines derived from patients who have undergone some form of chemotherapy84. Furthermore, the expression of SLFN11 was found to be directly regulated by an all-encompassing methylation pattern of the histones surrounding and inclusive of the SLFN11 gene body mediated by EZH2. In a set of SLFN11-low, previously treated SCLC cell lines, SLFN11 expression could be rescued through the administration of EZH2 inhibitors. Supporting the notions of a strong mesenchymal signature and chemoresistance, SLFN11 expression was found to be negatively correlated with the expression of TWIST1. This is yet another example of the effects of EZH2 hyperactivity in promoting the pervasiveness of SCLC through both acquired resistance and EMP.Emerging evidence has begun to show a strong correlation between a mesenchymal phenotype and chemoresistance. PARP1 has been shown to be highly upregulated in SCLCWhile numerous studies have highlighted the importance of EZH2 hyperactivity in SCLC development, they have only focused on one piece of the puzzle. Methyltransferases, such as EZH2, often become overexpressed in SCLC, making them attractive targets for therapeutic intervention. However, it is interesting to note that thus far, in SCLC patient samples, there are no documented copy number alterations of these proteins leading to their increased expression, suggesting that they are also subject to epigenetic control. Therefore, looking at a patient’s genetic profile will not indicate increased levels of EZH2 activity, but it will provide information about mutations occurring on the other side of the chromatin modification spectrum. There are numerous other chromatin modifiers that have a competitive relationship with PRC2, many of which are frequently mutated in SCLC. However, their role in SCLC progression remains significantly understudied. Therefore, the following section will summarize studies describing how frequently mutated chromatin modifiers contribute to cellular plasticity in the context of other cancers. These studies may provide important starting points to explore in SCLC.85, including SCLC. It works in concert with CREBBP to promote an open chromatin conformation through nucleosome sliding at competing sites with PRC2. The SWI/SNF complex is an ATP-dependent chromatin-remodeling complex that is made up of 15 subunits that form various combinations. SMARCA4/2 (BRG1/BRM1) and ARID1A/B (BAF250A/B) are integral subunits of the complex and are recurrently mutated in patient tumors in a mutually exclusive fashion86. While individual component mutations are less frequent (<5%) in SCLC, the mutations all result in SWI/SNF dysfunction. Thus, the compounded frequency (~20%) of the mutations provides a strong rationale for investigating the role of the SWI/SNF complex in SCLC. Especially given the antagonistic role of the SWI/SNF complex and PRC2, understanding the impact of SWI/SNF subunits may provide insight into therapeutic vulnerabilities of patient tumors harboring mutations related to EZH2 inhibitor resistance, as seen above, with CBP/P300 mutations increasing vulnerability to these inhibitors. SMARCA4 is also frequently mutated in NSCLC. Monitoring tumor formation in NSCLC GEMMs of lung adenocarcinoma  revealed that regardless of the cell type in which the alleles were targeted , Smarca4 loss led to increased metastasis87. In studies of both gastric cancer and pancreatic ductal adenocarcinoma (PDAC), analysis of patient samples and available cell lines revealed that loss of either Smarca4 or Arid1a led to increased metastasis and decreased CDH1 expression90. Furthermore, SMARCA4 loss in triple-negative breast cancer (TNBC) cell lines was associated with an increased mesenchymal signature, including increased expression of SNAIL, TWIST, and CDH2 and increased HIPPO pathway activity91. The HIPPO pathway is associated with an invasive phenotype92, and one of its key mediators is YAP1: a key non-NE marker found to be upregulated upon loss of ASCL1 and NEUROD1 in SCLC. In the study of TNBC, loss of SMARCA4 led to increased YAP signaling activity, and in a specific subtype (SL subtype: MDA-MB-468 cells), it was found to lead to increased expression of both YAP and its downstream targets. As previously described, the NE to non-NE transition in SCLC is frequently regulated by the chromatin modifiers LSD1 and KDM5A. The connection between the SCLC subtype and EMP signatures is gaining interest. Transcriptional analysis of cells isolated from RPM GEMMs identified gradual loss of NE marker expression  that coincided with increased expression of YAP1 and mesenchymal markers 93. Loss of the expression of these key SWI/SNF complex components may contribute to increased plasticity in SCLC through NE dedifferentiation and the accumulation of mesenchymal markers.The SWI/SNF chromatin-modifying complex is one of the most commonly mutated complexes across various cancer types94. Furthermore, in colon cancer, UTX was found to interact with CREBBP at the CDH1 promoter95. This interaction complements the inhibition of CDH1 transcription by PRC2 through increased methylation at the CDH1 promoter78. Together, these studies highlight the antagonistic relationship between UTX and PRC2. In a study evaluating the metastatic potential of breast cancer stem cells, UTX was found to work in concert with LSD1 and HDAC1 to remove the transcriptional activation signal H3K4me2 at the promoters of EMT-TFs, including SNAIL, ZEB1, and ZEB2, and thus inhibit their expression96. Interestingly, LSD1 localization at these promoters was found to be dependent on UTX expression. Given the contrasting levels of NE-high SCLC cells within primary tumors and NE-low cells within metastases, loss of UTX may be one of the molecular switches priming cells for EMT by increasing the expression of EMT-TFs.UTX (KDM6A) is a histone demethylase and is also found to be recurrently mutated in SCLC, but the impact of these mutations on SCLC development remains unexplored. UTX knockdown in NSCLC cells leads to a modest decrease in CDH1 expression73, we propose that loss-of-function mutations in chromatin modifiers  lead to loss of NE identity and increased plasticity 57. NK cells were found to be strictly excluded from mouse SCLC-N tumors58. Exploring the role of NK cells in tumor progression revealed that these cytotoxic cells did not have an apparent effect on primary tumor formation, but once activated, they were found to be key mediators controlling the metastatic spread of SCLC. Furthermore, NK cells identify their targets through recognition of NKG2DL. However, in SCLC, NKG2DL is transcriptionally silenced due in part to the cooperation of MYC and HDAC3. These findings highlight the role of chromatin modifiers as gatekeepers of the overlapping processes of immune recognition and metastatic spread in SCLC. Targeting these chromatin modifiers shows promising results in restoring the expression of immune markers and even recruiting immune cells to the SCLC TME  and is beginning to uncover critical interactions between tumor cells and the immune landscape of the TME through the creation and implementation of many physiologically relevant mouse models. Synthesis of these models along with knowledge gained from patient samples suggests that despite the substantial heterogeneity in the SCLC genome mutational profile, the mutations tend to be related to a few critical pathways that drive tumor development and progression. This review has focused on the emerging role of chromatin modifiers as the driving force behind key pathways promoting tumor development. Within mutant cells, an imbalance among chromatin modifiers  promotes immune evasion, increased plasticity, and the acquisition of mesenchymal characteristics Fig. . Further99. Other mediators of EZH2 localization at other target genes in SCLC remain unknown. It has been proposed that EZH2, and other Polycomb group (PcG) proteins, may be recruited to CpG islands through lncRNAs. In particular, HOTAIR was found to recruit EZH2 to the CDH1 promoter in nasopharyngeal carcinoma (NPC), leading to its transcriptional repression100. Given the increased expression of lncRNAs following CSE and the apparent hyperactivity of EZH2 in SCLC, exploring this relationship in the context of EZH2 localization to target genes in transformed SCLC cells may provide further insight into additional molecular vulnerabilities. Additionally, understanding the mechanisms of EZH2 localization may provide broader insight into the aberrant methylation landscape occurring during tumor progression across various cancers, including insight into how oncogenes tend to be selectively spared from hypermethylation to allow their persistent accessibility and expression. While its comprehensive role of EZH2 in SCLC has been appreciated in numerous studies, an understanding of the role of chromatin modifiers that antagonize EZH2 is lacking. These chromatin modifiers have been found to harbor recurrent mutations in SCLC and may represent therapeutic vulnerabilities. For example, loss of SMARCA4 in NSCLC cell lines led to sensitization to EZH2 inhibitors101. Understanding which patients may benefit most from therapy targeting epigenetic regulators may enhance the effectiveness of current immunotherapies60. Exploring the relationship among frequently altered chromatin modifiers will strengthen our understanding of the pathways exploited by transcriptional dysregulation during SCLC progression.Perhaps the most robust example of convergence despite such heterogeneity in SCLC is EZH2-mediated transcriptional dysregulation. EZH2 acts as a master regulator of SCLC plasticity. EZH2 hyperactivity in SCLC results in transcriptional reprogramming crucial for various aspects of both immune evasion and EMP in SCLC. It is responsible for silencing both MHC-I and CCL2, masking tumor cells from both the innate and adaptive arms of the immune system. As a regulator of lineage plasticity, EZH2 regulates the expression of CDH1 and interacts with lncRNAs to control the expression of various mesenchymal markers. EZH2 itself is subject to transcriptional regulation by NFIB. These examples show that EZH2 hyperactivity promotes SCLC tumor formation by affecting both the immunogenicity and plasticity of transforming cells. Extensive studies have compiled lists of EZH2 target genes; however, the mechanisms involved in EZH2 recruitment to target gene sites in the genome remain comparatively underexplored. In SCLC, it was discovered that the transcriptional repressor chromodomain Y-like (CDYL) recruits EZH2 to the cyclin-dependent kinase inhibitor 1 C (CDKN1C) promoter, leading to its silencing and further contributing to chemoresistance


Almost a half century ago, a group of nuclear proteins were co-purified with histones from calf thymus and termed as “high mobility group” (HMG) proteins because of their relative rapid mobility on SDS-PAGE gels . These HIn the first chapter , Ge et aIn the second chapter , Zhu et In the third chapter , WatanabIn the fourth chapter , Foglio In the fifth chapter , Yang etIn the sixth chapter , Saxena In the last chapter , Shih etIn summary, this special issue has provided comprehensive review of the intricate molecular mechanisms underlying the regulation of HMGB1 release and action by various PRRs as well as various endogenous molecules and exogenous pharmacological agents. It also contains thorough discussions of the pathogenic roles of extracellular HMGB1 in various inflammatory diseases such as sepsis, immune tolerance, ischemia/reperfusion injury, neuropathological pain, and perioperative neurocognitive disorders. Last but not the least, it provides an example of on-going investigations of the pathogenic role of HMGB1 in various inflammatory diseases. Therefore, this Special Issue will serve as an excellent reference for beginners to gain an comprehensive understanding of extensive HMGB1 research during the last 25 years.


The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads rapidly and harbors many mutations in the spike protein, but the origin of this virus variant remains unclear. We address the role of unusual virus evolution mechanisms such as hypermutation, out-of-frame reading, and recombination. Rather, regular Darwinian evolution, that is, the repeated selection of beneficial spike mutations, seems to have led to the appearance of the grossly altered spike protein of the Omicron variant. This virus unleashed a true pandemic in 2019 that continues until today, although a fair level of protection can be achieved by several newly developed vaccines and, hopefully, by herd immunity. Multiple SARS-CoV-2 variants of concern (VOCs) have been designated by the World Health Organization (WHO). Omicron (B.1.1.529) is the newest virus variant first reported from South Africa in November 2021 –6. The O–The origin of the Omicron variant remains unclear , 11. A cThe acquisition of many clustered mutations in the spike gene could in theory also be explained by more exotic evolutionary events that we will introduce and critically test. First, viral genomes can be subject to “hypermutation,” a process in which multiple changes occur in a clustered manner in parts of the viral genome. These mutations are introduced either by the viral polymerase, possibly by an error-prone mutant thereof, or by cellular restriction systems as exemplified by the HIV apolipoprotein B mRNA-editing catalytic polypeptide-like protein (APOBEC) case , 14. A cA second evolutionary scenario that could instantaneously generate many mutations is a double-frameshift event that causes the ribosome to temporarily read in a different reading frame . This ouTo broaden the test of the first two hypotheses, we also inspected the hypothetical evolutionary paths from any of the VOCs  to Omicron. This analysis also did not reveal any special mutational profile or frameshift events that could support these scenarios (results not shown).A third scenario that can trigger a gross yet sudden mutational event is recombination, the mixing of Omicron genome sequences with sequences derived from other viruses or the human host. Such swapping of genetic information is a well-known phenomenon in virology and is well documented for coronaviruses . For insHaving considered three unusual scenarios for the evolution of the Omicron variant, we cautiously conclude that regular virus evolution, that is, by the repeated selection of beneficial mutations in the spike gene or Darwinian evolution, underlies the appearance of this variant. Because Omicron first appeared in South Africa, it has been suggested that SARS-CoV-2 could evolve rapidly in this setting because of the weakened immune system of more than 20% of the local human population that is HIV infected . In lighThe most compelling evidence for this scenario of regular Darwinian evolution actually comes from inspection of the genetic changes, which reveals a profound preference for mutations that change the amino acid composition of the spike protein: 30 nonsilent changes versus 1 silent mutation . This evSome or all of these amino acid substitutions may affect the basic viral functions of receptor recognition and cell entry, and other substitutions may represent compensatory changes in the spike protein. We noticed a remarkable adjustment in the usage of charged amino acids: 2 positively charged residues were removed, but some 11 were gained, whereas a more balanced picture was seen for negatively charged residues (1 gained and 3 removed). Such changes in net or local charge could affect the interactions of the spike protein with other molecules, among which are the ACE2 receptor and antibodies raised by natural infection or immunization. Experimental studies are needed to test these ideas. Mutations in key domains of the spike protein may have an impact on the virulence and pathogenicity of this virus, possibly correlating with early epidemiological and clinical data showing that Omicron is less pathogenic than other SARS-CoV-2 variants .In conclusion, it seems likely that strong evolutionary pressure has led to the selection of the grossly altered spike protein of the Omicron variant. However, much remains currently unknown. First of all, extensive sampling may reveal virus isolates that are intermediate between the Wuhan and Omicron strains, which could more firmly prove or deny the exact path of virus evolution. Experiments should then address the biological effects imposed by the many spike protein substitutions, both direct effects on virus replication and also indirect effects through escape from host-induced immune pressure. In the meantime, we should rapidly update the RNA-based vaccines against this rapidly spreading SARS-CoV-2 variant.