[Correlation between common driver gene variations and clinicopathological typing in lung adenocarcinoma].

Objective: To correlate the common driver gene variations in primary lung adenocarcinoma with their clinical characteristics and histopathological subtypes. Methods: There were 4 995 cases of primary lung adenocarcinoma diagnosed at Weifang People's Hospital of Shandong Province from January 2015 to December 2021 which were retrospectively analyzed. Among them 1 983 cases were evaluated for their histopathological subtype; 3 012 were analyzed for the correlation of their histopathological subtypes and corresponding driver gene variations, including invasive non-mucinous adenocarcinoma (INMA) and invasive mucinous adenocarcinoma (IMA), and morphologically, poorly-differentiated, moderately-differentiated and well-differentiated adenocarcinomas. Next-generation sequencing was used to detect variations in EGFR, KRAS, ALK, RET, ROS1, MET, HER2, or BRAF driver genes. Results: There were 2 384 males and 2 611 females. EGFR and ALK variations were more commonly found in female patients aged 60 years or older, with EGFR mutation rate in clinical stage Ⅰ (25.80%) significantly higher than in other stages (P<0.05). KRAS mutations were more commonly detected in male smokers aged 60 years or older, HER2 mutations were more commonly in patients younger than 60 years, and RET mutations were more commonly in non-smokers (all P<0.05). No correlation was found between ROS1, MET, and BRAF gene variations and their clinical characteristics (P>0.05). For the histopathological subtypes, among the 1 899 cases of acinar adenocarcinoma, EGFR mutation rate was the highest (67.30%) compared to the other genes. Exon 21 L858R and exon 19 del were the main mutation sites in IMA and INMA, with a higher mutation rate at exon 20 T790M (11.63%) in micropapillary adenocarcinoma. In IMA, KRAS had the highest overall mutation rate (43.80%), with statistically significant difference in mutation rates of exon 2 G12D and exon 2 G12V in acinar adenocarcinoma, solid, and IMA (P<0.05). KRAS mutation at various sites were higher in poorly differentiated groups compared to moderately- and well-differentiated groups (P<0.05). HER2 mutations were more commonly observed in acinar adenocarcinoma, papillary, and micropapillary adenocarcinoma of INMA. BRAF mutation was higher in micropapillary adenocarcinoma compared with other types (P<0.05). Conclusions: Variations in EGFR, ALK, KRAS, HER2, and RET in primary lung adenocarcinoma are associated with patients' age, smoking history, and clinical stage, and driver gene mutations vary among different histopathological subtypes. EGFR mutations are predominant in INMA, while KRAS mutations are predominant in IMA.

In silico identification of a novel Cdc2-like kinase 2 (CLK2) inhibitor in triple negative breast cancer.

Dysregulation of RNA splicing processes is intricately linked to tumorigenesis in various cancers, especially breast cancer. Cdc2-like kinase 2 (CLK2), an oncogenic RNA-splicing kinase pivotal in breast cancer, plays a significant role, particularly in the context of triple-negative breast cancer (TNBC), a subtype marked by substantial medical challenges due to its low survival rates. In this study, we employed a structure-based virtual screening (SBVS) method to identify potential CLK2 inhibitors with novel chemical structures for treating TNBC. Compound 670551 emerged as a novel CLK2 inhibitor with a 50% inhibitory concentration (IC50) value of 619.7 nM. Importantly, Compound 670551 exhibited high selectivity for CLK2 over other protein kinases. Functionally, this compound significantly reduced the survival and proliferation of TNBC cells. Results from a cell-based assay demonstrated that this inhibitor led to a decrease in RNA splicing proteins, such as SRSF4 and SRSF6, resulting in cell apoptosis. In summary, we identified a novel CLK2 inhibitor as a promising potential treatment for TNBC therapy.

CLK2 mediates IκBα-independent early termination of NF-κB activation by inducing cytoplasmic redistribution and degradation.

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.

The anticancer potential of the CLK kinases inhibitors 1C8 and GPS167 revealed by their impact on the epithelial-mesenchymal transition and the antiviral immune response.

The diheteroarylamide-based compound 1C8 and the aminothiazole carboxamide-related compound GPS167 inhibit the CLK kinases, and affect the proliferation of a broad range of cancer cell lines. A chemogenomic screen previously performed with GPS167 revealed that the depletion of components associated with mitotic spindle assembly altered sensitivity to GPS167. Here, a similar screen performed with 1C8 also established the impact of components involved in mitotic spindle assembly. Accordingly, transcriptome analyses of cells treated with 1C8 and GPS167 indicated that the expression and RNA splicing of transcripts encoding mitotic spindle assembly components were affected. The functional relevance of the microtubule connection was confirmed by showing that subtoxic concentrations of drugs affecting mitotic spindle assembly increased sensitivity to GPS167. 1C8 and GPS167 impacted the expression and splicing of transcripts in pathways relevant to tumor progression, including MYC targets and the epithelial mesenchymal transition (EMT). Finally, 1C8 and GPS167 altered the expression and alternative splicing of transcripts involved in the antiviral immune response. Consistent with this observation, depleting the double-stranded RNA sensor DHX33 suppressed GPS167-mediated cytotoxicity on HCT116 cells. Our study uncovered molecular mechanisms through which 1C8 and GPS167 affect cancer cell proliferation as well as processes critical for metastasis.

Csk restrains BCR-mediated ROS production and contributes to germinal center selection and affinity maturation.

Compared with naïve B cells, the B cell receptor (BCR) signal in germinal center (GC) B cells is attenuated; however, the significance of this signaling attenuation has not been well defined. Here, to investigate the role of attenuation of BCR signaling, we employed a Csk mutant mouse model in which Csk deficiency in GC B cells resulted in augmentation of net BCR signaling with no apparent effect on antigen presentation. We found that Csk is required for GC maintenance and efficient antibody affinity maturation. Mechanistically, ROS-induced apoptosis was exacerbated concomitantly with mitochondrial dysfunction in Csk-deficient GC B cells. Hence, our data suggest that attenuation of the BCR signal restrains hyper-ROS production, thereby protecting GC B cells from apoptosis and contributing to efficient affinity maturation.

Impact of Abl2/Arg deficiency on anxiety and depressive behaviors in mice.

Abl2/Arg (ABL-related gene) is a member of the Abelson family of nonreceptor tyrosine kinases, known for its role in tumor progression, metastasis, tissue injury responses, inflammation, neural degeneration, and other diseases. In this study, we developed Abl2/Arg knockout (abl2-/-) mice to explore its impact on sensory/motor functions and emotion-related behaviors. Our findings show that abl2-/- mice exhibit normal growth and phenotypic characteristics, closely resembling their wild-type (WT) counterparts. Behavioral tests, including the elevated plus maze, marble-burying behavior test, and open field test, indicated pronounced anxiety-like behaviors in abl2-/- mice compared to WT mice. Furthermore, in the tail suspension test, abl2-/- mice showed a significant decrease in mobility time, suggesting depressive-like behavior. Conversely, in the Y-maze and cliff avoidance reaction tests, no notable differences were observed between abl2-/- and WT mice, suggesting the absence of working memory deficits and impulsivity in abl2-/- mice. Proteomic analysis of the hippocampus in abl2-/- mice highlighted significant alterations in proteins related to anxiety and depression, especially those associated with the GABAergic synapse in inhibitory neurotransmission. The expression of Gabbr2 was significantly reduced in the hippocampus of abl2-/- compared to WT mice, and intraperitoneal treatment of GABA receptor agonist Gaboxadol normalized anxiety/depression-related behaviors of abl2-/- mice. These findings underscore the potential role of Abl2/Arg in influencing anxiety and depressive-like behaviors, thereby contributing valuable insights into its broader physiological and pathological functions.

CircCOX6A1 suppresses osteogenic differentiation and aggravates osteoporosis via miR-512-3p/DYRK2 axis.

BACKGROUND: Osteoporosis (OP), characterized by compromised bone integrity and increased fracture risk, poses a significant health challenge. Circular RNAs (circRNAs) have emerged as crucial regulators in various pathophysiological processes, prompting investigation into their role in osteoporosis. This study aimed to elucidate the involvement of circCOX6A1 in OP progression and understand its underlying molecular mechanisms. The primary objective was to explore the impact of circCOX6A1 on bone marrow-derived mesenchymal stem cells (BMSCs) and its potential interactions with miR-512-3p and DYRK2. METHODS: GSE161361 microarray analysis was employed to assess circCOX6A1 expression in OP patients. We utilized in vitro and in vivo models, including BMSC cultures, osteogenic differentiation assays, and an OVX-induced mouse model of OP. Molecular techniques such as quantitative RT-PCR, western blotting, and functional assays like alizarin red staining (ARS) were employed to evaluate circCOX6A1 effects on BMSC proliferation, apoptosis, and osteogenic differentiation. The interaction between circCOX6A1, miR-512-3p, and DYRK2 was investigated through dual luciferase reporter assays, RNA immunoprecipitation, and RNA pull-down assays. RESULTS: CircCOX6A1 was found to be upregulated in osteoporosis patients, and its expression inversely correlated with osteogenic differentiation of BMSCs. CircCOX6A1 knockdown enhanced osteogenic differentiation, as evidenced by increased mineralized nodule formation and upregulation of osteogenic markers. In vivo, circCOX6A1 knockdown ameliorated osteoporosis progression in OVX mice. Mechanistically, circCOX6A1 acted as a sponge for miR-512-3p, subsequently regulating DYRK2 expression. CONCLUSION: This study provides compelling evidence for the role of circCOX6A1 in osteoporosis pathogenesis. CircCOX6A1 negatively regulates BMSC osteogenic differentiation through the miR-512-3p/DYRK2 axis, suggesting its potential as a therapeutic target for mitigating OP progression.

Prenatal treatment with preimplantation factor improves early postnatal neurogenesis and cognitive impairments in a mouse model of Down syndrome.

Down syndrome (DS) is a genetic disease characterized by a supernumerary chromosome 21. Intellectual deficiency (ID) is one of the most prominent features of DS. Central nervous system defects lead to learning disabilities, motor and language delays, and memory impairments. At present, a prenatal treatment for the ID in DS is lacking. Subcutaneous administration of synthetic preimplantation factor (sPIF, a peptide with a range of biological functions) in a model of severe brain damage has shown neuroprotective and anti-inflammatory properties by directly targeting neurons and microglia. Here, we evaluated the effect of PIF administration during gestation and until weaning on Dp(16)1Yey mice (a mouse model of DS). Possible effects at the juvenile stage were assessed using behavioral tests and molecular and histological analyses of the brain. To test the influence of perinatal sPIF treatment at the adult stage, hippocampus-dependent memory was evaluated on postnatal day 90. Dp(16)1Yey pups showed significant behavioral impairment, with impaired neurogenesis, microglial cell activation and a low microglial cell count, and the deregulated expression of genes linked to neuroinflammation and cell cycle regulation. Treatment with sPIF restored early postnatal hippocampal neurogenesis, with beneficial effects on astrocytes, microglia, inflammation, and cell cycle markers. Moreover, treatment with sPIF restored the level of DYRK1A, a protein that is involved in cognitive impairments in DS. In line with the beneficial effects on neurogenesis, perinatal treatment with sPIF was associated with an improvement in working memory in adult Dp(16)1Yey mice. Perinatal treatment with sPIF might be an option for mitigating cognitive impairments in people with DS.

Comparison of diagnostic performance between Oncomine Dx target test and AmoyDx panel for detecting actionable mutations in lung cancer.

Companion diagnostic (CDx) tests play important roles in identifying oncogenic driver genes and tailoring effective molecularly targeted therapies for lung cancer patients. In Japan, the Oncomine Dx target test (ODxTT) and the AmoyDx pan lung cancer PCR panel (AmoyDx) are prominent CDx tests and only one of these tests is covered by the domestic insurance system. However, these CDx tests cover different target regions and apply different technologies (ODxTT is amplicon-based next-generation sequencing and AmoyDx is multiplex PCR-based assay), which may lead to missing of actionable mutations affecting patient prognosis. Here, we performed a direct comparison analysis of 1059 genetic alterations of eight driver genes from 131 samples and evaluated the concordance between two CDx tests for detecting actionable variants and fusions. When excluding the eight uncovered variants (ODxTT: two variants, AmoyDx: six variants), the overall percent agreement was 97.6% (1026/1051) with 89.0% of overall positive percent agreement (89/100) and 98.5% of overall negative percent agreement (937/951). Of the 25 discordant genetic alterations, two were undetected despite being covered in the AmoyDx (one EGFR variant and one ROS1 fusion). Furthermore, there were potential false positives in the ODxTT (nine MET exon 14 skippings) and in the AmoyDx (five variants, six ROS1 and three RET fusions). These potential false positives in the AmoyDx likely due to non-specific amplification, which was validated by the unique molecular barcoding sequencing. The ODxTT missed two uncovered EGFR rare variants, which was visually confirmed in the raw sequencing data. Our study provides insights into real-world performance of CDx tests for lung cancer and ensures reliability to advance precision medicine.

Efficacy and safety of crizotinib in the treatment of advanced non-small cell lung cancer with ROS1 gene fusion: a systematic literature review and meta-analysis of real-world evidence.

BACKGROUND: Crizotinib was approved to treat patients with advanced non-small cell lung cancer (aNSCLC) with ROS proto-oncogene 1 (ROS1) gene fusion in 2016. We conducted a systematic literature review to identify real-world evidence (RWE) studies and estimated the efficacy and safety of crizotinib using meta-analyses (MA) for objective response rate (ORR), real-world progression-free survival (PFS), and overall survival (OS). METHODS: We searched MEDLINE®, Embase, and Cochrane CENTRAL from January 2016 to March 2023 using Ovid® for published single-arm or comparative RWE studies evaluating patients (N ≥ 20) receiving crizotinib monotherapy for aNSCLC with ROS1 gene fusion. Pooled estimates for ORR and grade 3/4 adverse events (AEs) were derived using the metafor package in R while pooled estimates for median real-world PFS (rwPFS) and OS were derived using reconstructed individual patient data from published Kaplan-Meier curves. The primary analysis included all studies regardless of crizotinib line of therapy; a subgroup analysis (SA) was conducted using studies evaluating patients receiving first-line crizotinib. RESULTS: Fourteen studies met the eligibility criteria and were considered feasible for MA. For the primary analysis, the pooled ORR (N = 9 studies) was 70.6 % (95 % confidence interval [CI]: 57.0, 81.3), median rwPFS was 14.5 months (N = 11 studies), and OS was 40.2 months (N = 9 studies). In the SA, the pooled ORR (N = 4 studies) was 81.1 % (95 % CI: 76.1, 85.2) and the median rwPFS (N = 4 studies) and OS (N = 2 studies) were 18.1 and 60 months, respectively. All MAs were associated with significant heterogeneity (I2 > 25 %). Grade 3/4 AEs occurred in 18.7 % of patients (pooled estimate). CONCLUSION: The results from this study are consistent with clinical trial data and, taken collectively, supports crizotinib as a safe and effective treatment across different lines of therapy in patients with ROS1 aNSCLC in the real-world setting.

Clinical treatment patterns, molecular characteristics and survival outcomes of ROS1-rearranged non-small cell lung cancer: A large multicenter retrospective study.

BACKGROUND: Non-small cell lung cancer (NSCLC) harboring ROS1 rearrangements is a molecular subset that exhibits favorable responses to tyrosine kinase inhibitor (TKI) treatment than chemotherapy. This study investigated real-world treatment patterns and survival outcomes among patients with ROS1-rearranged advanced NSCLC. METHODS: We conducted a retrospective analysis of patients with ROS1-rearranged advanced NSCLC treated in four different hospitals in China from August 2018 to March 2022. The study analyzed gene fusion distribution, resistance patterns, and survival outcomes. RESULTS: ROS1 rearrangement occurs in 1.8 % (550/31,225) of our study cohort. CD74 was the most common ROS1 fusion partner, accounting for 45.8 %. Crizotinib was used in 73.9 % of patients in the first-line treatment, and an increased use of chemotherapy, ceritinib, and lorlatinib was seen in the second-line setting. Lung (43.2 %) and brain (27.6 %) were the most common sites of progression in first-line setting, while brain progression (39.2 %) was the most common site of progression in second-line. Median overall survival was 46 months (95 % confidence intervals: 39.6-52.4). First-line crizotinib use yielded significantly superior survival outcomes over chemotherapy in terms of progression-free (18.5 vs. 6.0; p < 0.001) and overall survival (49.8 vs. 37; p = 0.024). The choice of treatment in the latter line also had survival implications, wherein survival outcomes were better when first-line crizotinib was followed by sequential TKI therapy than first-line chemotherapy followed by TKI therapy. CONCLUSIONS: Our study provided insights into the real-world treatment, drug resistance patterns, and survival outcomes among patients with ROS1-rearranged NSCLC. This information serves as a valuable reference for guiding the treatment of this molecular subset of NSCLC.

Reclassification of a spindle cell sarcoma after identification of a TFG-ROS1 fusion: A case demonstrating the clinical benefit of next-generation sequencing in sarcoma.

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) are rare mesenchymal soft tissue sarcomas that often present diagnostic challenges due to their wide and varied morphology. A subset of IMTs have fusions involving ALK or ROS1. The role of next-generation sequencing (NGS) for classification of unselected sarcomas remains controversial. METHODS AND RESULTS: We report a case of a metastatic sarcoma in a 34-year-old female originally diagnosed as an unclassified spindle cell sarcoma with myofibroblastic differentiation and later reclassified as IMT after NGS revealed a TFG-ROS1 rearrangement. Histologically, the neoplasm had spindle cell morphology with a lobulated to focally infiltrative growth pattern with scant inflammatory cell infiltrate. Immunohistochemistry demonstrated focal desmin and variable smooth muscle actin staining but was negative for SOX10, S100, and CD34. Fluorescence in situ hybridization was negative for USP6 or ALK gene rearrangements. NGS revealed a TFG-ROS1 rearrangement and the patient was treated with crizotinib with clinical benefit. CONCLUSIONS: We discuss the role of NGS as well as its potential benefit in patients with unresectable, ALK-negative metastatic disease. Considering this case and previous literature, we support the use of NGS for patients requiring systemic treatment.

Targeted therapeutic options in early and metastatic NSCLC-overview.

The complex therapeutic strategy of non-small cell lung cancer (NSCLC) has changed significantly in recent years. Disease-free survival increased significantly with immunotherapy and chemotherapy registered in perioperative treatments, as well as adjuvant registered immunotherapy and targeted therapy (osimertinib) in case of EGFR mutation. In oncogenic-addictive metastatic NSCLC, primarily in adenocarcinoma, the range of targeted therapies is expanding, with which the expected overall survival increases significantly, measured in years. By 2021, the FDA and EMA have approved targeted agents to inhibit EGFR activating mutations, T790 M resistance mutation, BRAF V600E mutation, ALK, ROS1, NTRK and RET fusion. In 2022, the range of authorized target therapies was expanded. With therapies that inhibit KRASG12C, EGFR exon 20, HER2 and MET. Until now, there was no registered targeted therapy for the KRAS mutations, which affect 30% of adenocarcinomas. Thus, the greatest expectation surrounded the inhibition of the KRAS G12C mutation, which occurs in ∼15% of NSCLC, mainly in smokers and is characterized by a poor prognosis. Sotorasib and adagrasib are approved as second-line agents after at least one prior course of chemotherapy and/or immunotherapy. Adagrasib in first-line combination with pembrolizumab immunotherapy proved more beneficial, especially in patients with high expression of PD-L1. In EGFR exon 20 insertion mutation of lung adenocarcinoma, amivantanab was registered for progression after platinum-based chemotherapy. Lung adenocarcinoma carries an EGFR exon 20, HER2 insertion mutation in 2%, for which the first targeted therapy is trastuzumab deruxtecan, in patients already treated with platinum-based chemotherapy. Two orally administered selective c-MET inhibitors, capmatinib and tepotinib, were also approved after chemotherapy in adenocarcinoma carrying MET exon 14 skipping mutations of about 3%. Incorporating reflex testing with next-generation sequencing (NGS) expands personalized therapies by identifying guideline-recommended molecular alterations.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

AR-V7 expression facilitates accelerated G2/M phase transition in castration-resistant prostate cancer.

The emergence of AR-V7, a truncated isoform of AR upon androgen deprivation therapy treatment, leads to the development of castration resistant prostate cancer (CRPC). Understanding mechanisms that regulate AR-V7 expression is critical for developing newer therapeutic strategies. In this study, we have investigated the regulation of AR-V7 during cell cycle and identified a distinct pattern of periodic fluctuation, peaking during G2/M phase. This fluctuation correlates with the expression of Cdc-2 like kinase 1 (CLK1) and phosphorylated serine/arginine-rich splicing factor 1 (p-SRSF1) during these phases, pointing towards their role in AR-V7 generation. Functional assays reveal that CLK1 knockdown prolongs the S phase, leading to altered cell cycle distribution and increased accumulation of AR-V7 and pSRSF1 in G1/S phase. Conversely, CLK1 overexpression rescues AR-V7 and p-SRSF1 levels in the G2/M phase, consistent with observed cell cycle alterations upon AR-V7 knockdown and overexpression in CRPC cells. Furthermore, overexpression of kinase-deficient CLK1 mutant leads to diminished AR-V7 levels during G2/M, underlining the essential contribution of CLK1's kinase activity in modulating AR-V7 expression. Collectively, our findings, for the first time, show periodic regulation of AR-V7 expression, its effect on cell cycle progression and the critical role of CLK1-pSRSF1 axis in modulating AR-V7 expression throughout the cell cycle.

The TRAF3-DYRK1A-RAD54L2 complex maintains ACE2 expression to promote SARS-CoV-2 infection.

Angiotensin converting enzyme 2 (ACE2), the host receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is differentially expressed in a wide variety of tissues and cell types. The expression of ACE2 is under tight regulation, but the mechanisms regulating ACE2 expression have not yet been well defined. Through a genome-wide CRISPR knockout screen, we discovered that host factors TRAF3, DYRK1A, and RAD54L2 (TDR) form a complex to regulate the expression of ACE2. Knockout of TRAF3, DYRK1A, or RAD54L2 reduces the mRNA levels of ACE2 and inhibits the cellular entry of SARS-CoV-2. On the other hand, SARS-CoV-2 continuously evolves by genetic mutations for the adaption to the host. We have identified mutations in spike (S) (P1079T) and nucleocapsid (N) (S194L) that enhance the replication of SARS-CoV-2 in cells that express ACE2 at a low level. Our results have revealed the mechanisms for the transcriptional regulation of ACE2 and the adaption of SARS-CoV-2. IMPORTANCE: The expression of ACE2 is essential for the entry of SARS-CoV-2 into host cells. We identify a new complex-the TDR complex-that acts to maintain the abundance of ACE2 in host cells. The identification and characterization of the TDR complex provide new targets for the development of therapeutics against SARS-CoV-2 infection. By analysis of SARS-CoV-2 virus replicating in cells expressing low levels of ACE2, we identified mutations in spike (P1079T) and nucleocapsid (S194L) that overcome the restriction of limited ACE2. Functional analysis of these key amino acids in S and N extends our knowledge of the impact of SARS-CoV-2 variants on virus infection and transmission.