Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells.

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.

Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression.

Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.

Effective Cycle Slip Detection and Repair for PPP/INS Integrated Systems.

Cycle slip (CS) is a primary error source in Precise Point Positioning/Inertial Navigation System (PPP/INS) integrated systems. In this study, an INS-aided CS detection and repair method is presented. It utilizes high-precision INS information instead of a pseudorange to remove the satellite⁻receiver geometric range in the wide-lane (WL) and ionospheric-free (IF) phase combinations and creates an INS-aided WL (WL-INS) model and an INS-aided IF (IF-INS) model. Since INS information is superior to pseudorange, the INS-aided models have high detection accuracy. However, the effectiveness of INS-aided models cannot persist for a long time because of INS accumulation error. To overcome the disturbance of INS error, improved INS-aided models are proposed. This idea takes advantage of the long wavelength of WL combination and tries to fix WL CS. Once it succeeds, the INS error can be evaluated and removed. The proposed method was tested using land vehicle data, in which simulated cycle slips and signal interruption were introduced. The results show that this method can accurately detect and repair different cycle slips between the continuous Global Positioning System (GPS) epoch. When it comes to the cycle slip after a GPS interruption, the method can also accelerate PPP re-convergence, as it is not affected by the inertial accumulation error.

The Notch Ligand DLL4 Defines a Capability of Human Dendritic Cells in Regulating Th1 and Th17 Differentiation.

Notch signaling regulates multiple helper CD4(+) T cell programs. We have recently demonstrated that dendritic cells (DCs) expressing the Notch ligand DLL4 are critical for eliciting alloreactive T cell responses and induction of graft-versus-host disease in mice. However, the human counterpart of murine DLL4(+) DCs has yet to be examined. We report the identification of human DLL4(+) DCs and their critical role in regulating Th1 and Th17 differentiation. CD1c(+) DCs and plasmacytoid DCs (pDCs) from the peripheral blood (PB) of healthy donors did not express DLL4. In contrast, patients undergoing allogeneic hematopoietic stem cell transplantation had a 16-fold more DLL4(+)CD1c(+) DCs than healthy donors. Upon activation of TLR signaling, healthy donor-derived CD1c(+) DCs dramatically upregulated DLL4, as did pDCs to a lesser extent. Activated DLL4(+) DCs were better able to promote Th1 and Th17 differentiation than unstimulated PB DCs. Blocking DLL4 using a neutralizing Ab decreased Notch signaling in T cells stimulated with DLL4(+) DCs, and it reduced the generation of Th1 and Th17 cells. Both NF-κB and STAT3 were crucial for inducing DLL4 in human DCs. Interestingly, STAT3 directly activated DLL4 transcription and inhibiting STAT3 alone was sufficient to reduce DLL4 in activated PB DCs. Thus, DLL4 is a unique functional molecule of human circulating DCs critical for directing Th1 and Th17 differentiation. These findings identify a pathway for therapeutic intervention for inflammatory disorders in humans, such as graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, autoimmunity, and tumor immunity.

Ezh2 regulates transcriptional and posttranslational expression of T-bet and promotes Th1 cell responses mediating aplastic anemia in mice.

Acquired aplastic anemia (AA) is a potentially fatal bone marrow (BM) failure syndrome. IFN-γ-producing Th1 CD4(+) T cells mediate the immune destruction of hematopoietic cells, and they are central to the pathogenesis. However, the molecular events that control the development of BM-destructive Th1 cells remain largely unknown. Ezh2 is a chromatin-modifying enzyme that regulates multiple cellular processes primarily by silencing gene expression. We recently reported that Ezh2 is crucial for inflammatory T cell responses after allogeneic BM transplantation. To elucidate whether Ezh2 mediates pathogenic Th1 responses in AA and the mechanism of Ezh2 action in regulating Th1 cells, we studied the effects of Ezh2 inhibition in CD4(+) T cells using a mouse model of human AA. Conditionally deleting Ezh2 in mature T cells dramatically reduced the production of BM-destructive Th1 cells in vivo, decreased BM-infiltrating Th1 cells, and rescued mice from BM failure. Ezh2 inhibition resulted in significant decrease in the expression of Tbx21 and Stat4, which encode transcription factors T-bet and STAT4, respectively. Introduction of T-bet but not STAT4 into Ezh2-deficient T cells fully rescued their differentiation into Th1 cells mediating AA. Ezh2 bound to the Tbx21 promoter in Th1 cells and directly activated Tbx21 transcription. Unexpectedly, Ezh2 was also required to prevent proteasome-mediated degradation of T-bet protein in Th1 cells. Our results demonstrate that Ezh2 promotes the generation of BM-destructive Th1 cells through a mechanism of transcriptional and posttranscriptional regulation of T-bet. These results also highlight the therapeutic potential of Ezh2 inhibition in reducing AA and other autoimmune diseases.

Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL.

A notable number of acute lymphoblastic leukemia (ALL) patients develop CD19-positive relapse within 1 year after receiving chimeric antigen receptor (CAR) T cell therapy. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes. Here, we present 101,326 single-cell transcriptomes and surface protein landscape from the infusion products of 12 ALL patients. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of T helper 2 function was associated with CD19-positive relapse compared with durable responders (remission, >54 months). Proteomic data revealed that the frequency of early memory T cells, rather than activation or coinhibitory signatures, could distinguish the relapse. These findings were corroborated by independent functional profiling of 49 patients, and an integrative model was developed to predict the response. Our data unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long-term remission.

Identification and evaluation of the role of the manganese efflux protein in Deinococcus radiodurans.

BACKGROUND: Deinococcus radiodurans accumulates high levels of manganese ions, and this is believed to be correlated with the radiation resistance ability of this microorganism. However, the maintenance of manganese ion homeostasis in D. radiodurans remains to be investigated. RESULTS: In this study, we identified the manganese efflux protein (MntE) in D. radiodurans. The null mutant of mntE was more sensitive than the wild-type strain to manganese ions, and the growth of the mntE mutant was delayed in manganese-supplemented media. Furthermore, there was a substantial increase in the in vivo concentration of manganese ions. Consistent with these characteristics, the mntE mutant was more resistant to H2O2, ultraviolet rays, and γ-radiation. The intracellular protein oxidation (carbonylation) level of the mutant strain was remarkably lower than that of the wild-type strain. CONCLUSIONS: Our results indicated that dr1236 is indeed a mntE homologue and is indispensable for maintaining manganese homeostasis in D. radiodurans. The data also provide additional evidence for the involvement of intracellular manganese ions in the radiation resistance of D. radiodurans.

Smooth muscle SIRT1 reprograms endothelial cells to suppress angiogenesis after ischemia.

Objective: Vascular smooth muscle cells (VSMCs) undergo the phenotypic changes from contractile to synthetic state during vascular remodeling after ischemia. SIRT1 protects against stress-induced vascular remodeling via maintaining VSMC differentiated phenotype. However, the effect of smooth muscle SIRT1 on the functions of endothelial cells (ECs) has not been well clarified. Here, we explored the role of smooth muscle SIRT1 in endothelial angiogenesis after ischemia and the underlying mechanisms. Methods: We performed a femoral artery ligation model using VSMC specific human SIRT1 transgenic (SIRT1-Tg) and knockout (KO) mice. Angiogenesis was assessed in in vivo by quantification of the total number of capillaries, wound healing and matrigel plug assays, and in vitro ECs by tube formation, proliferation and migration assays. The interaction of HIF1α with circRNA was examined by using RNA immunoprecipitation, RNA pull-down and in situ hybridization assays. Results: The blood flow recovery was significantly attenuated in SIRT1-Tg mice, and markedly improved in SIRT1-Tg mice treated with SIRT1 inhibitor EX527 and in SIRT1-KO mice. The density of capillaries significantly decreased in the ischemic gastrocnemius of SIRT1-Tg mice compared with SIRT1-KO and WT mice, with reduced expression of VEGFA, which resulted in decreased number of arterioles. We identified that the phenotypic switching of SIRT1-Tg VSMCs was attenuated in response to hypoxia, with high levels of contractile proteins and reduced expression of the synthetic markers and NG2, compared with SIRT1-KO and WT VSMCs. Mechanistically, SIRT1-Tg VSMCs inhibited endothelial angiogenic activity induced by hypoxia via the exosome cZFP609. The cZFP609 was delivered into ECs, and detained HIF1α in the cytoplasm via its interaction with HIF1α, thereby inhibiting VEGFA expression and endothelial angiogenic functions. Meantime, the high cZFP609 expression was observed in the plasma of the patients with atherosclerotic or diabetic lower extremity peripheral artery disease, associated with reduced ankle-brachial index. Knockdown of cZFP609 improved blood flow recovery after hindlimb ischemia in SIRT1-Tg mice. Conclusions: Our findings demonstrate that SIRT1 may impair the plasticity of VSMCs. cZFP609 mediates VSMCs to reprogram endothelial functions, and serves as a valuable indicator to assess the prognosis and clinical outcomes of ischemic diseases.

Melatonin protects against chromium(VI)-induced cardiac injury via activating the AMPK/Nrf2 pathway.

Chromium (Cr) threatens health by causing oxidative stress. However, effective therapy for cardiac damage mediated by potassium dichromate (K2Cr2O7) still has not been defined. Melatonin (MT) possesses a number of biological activities. Our study was performed to explore the effect and mechanism of MT on Cr(VI)-induced cardiac damage by conducting both in vitro and in vivo studies. Twenty eight male Wistar rats were randomly assigned to four groups: control, MT (20 mg/kg subcutaneously), K2Cr2O7 (4 mg/kg intraperitoneally), and K2Cr2O7 + MT. We measured biomarkers of oxidative stress and cardiac function, and performed histopathological analysis, assay of terminal deoxynucleotidyl transferase-mediated deoxyuracil nucleoside triphosphate nick end labeling and protein levels, and the viability assay of cultured cardiomyocytes in vitro. Our results showed that MT ameliorated K2Cr2O7-induced oxidative stress, apoptosis, and the release of inflammatory mediators in the rat heart. MT also promoted adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, upregulated expression of proteins that nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1, and nicotinamide adenine dinucleotide phosphatase: quinone-acceptor 1, and inhibited nuclear factor kappa B in the heart of rats exposed to K2Cr2O7. Furthermore, MT increased B-cell lymphoma gene 2 (Bcl-2) and B-cell lymphoma extra large protein levels and decreased cleaved caspase 3, P53, and Bcl-2-associated X protein levels. Furthermore, the experiment in vitro showed that MT increased the cells viability and protein levels of Nrf2 and phosphorylated-AMPK in H9C2 cells treated with K2Cr2O7. Collectively, our results demonstrate that MT protects against Cr-induced cardiac damage via activating the AMPK/Nrf2 pathway.

Chimeric-Antigen-Receptor (CAR) T Cells and the Factors Influencing their Therapeutic Efficacy.

Immunotherapeutic treatments for malignant cancers have revolutionized the medical and scientific fields. Lymphocytes engineered to display chimeric antigen receptor (CAR) molecules contribute to the exciting advancements that have stemmed from a greater understanding of cell structure and function, biological interactions, and the unique tumor microenvironment. CAR T cells circumvent the unique immune evasion capability of tumors by acting in a major histocompatibility complex (MHC) independent manner. Various factors contribute to the efficacy of CAR therapy, including CAR structure, gene transfer strategies, in vitro culture system, target selection, and preconditioning regimens. While recent clinical trials have shown promising success, cytotoxicity and other various challenges need to be addressed before CAR therapy can reach its full clinical potency. This review will discuss factors associated with CAR therapeutic success and the difficulties that continue to be a focus of research around the world.

Ezh2 phosphorylation state determines its capacity to maintain CD8+ T memory precursors for antitumor immunity.

Memory T cells sustain effector T-cell production while self-renewing in reaction to persistent antigen; yet, excessive expansion reduces memory potential and impairs antitumor immunity. Epigenetic mechanisms are thought to be important for balancing effector and memory differentiation; however, the epigenetic regulator(s) underpinning this process remains unknown. Herein, we show that the histone methyltransferase Ezh2 controls CD8+ T memory precursor formation and antitumor activity. Ezh2 activates Id3 while silencing Id2, Prdm1 and Eomes, promoting the expansion of memory precursor cells and their differentiation into functional memory cells. Akt activation phosphorylates Ezh2 and decreases its control of these transcriptional programs, causing enhanced effector differentiation at the expense of T memory precursors. Engineering T cells with an Akt-insensitive Ezh2 mutant markedly improves their memory potential and capability of controlling tumor growth compared to transiently inhibiting Akt. These findings establish Akt-mediated phosphorylation of Ezh2 as a critical target to potentiate antitumor immunotherapeutic strategies.

Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination.

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.

Extracellular dGMP enhances Deinococcus radiodurans tolerance to oxidative stress.

Free extracellular DNA provides nutrition to bacteria and promotes bacterial evolution by inducing excessive mutagenesis of the genome. To understand the influence of extracellular DNA fragments on D. radiodurans, we investigated cell growth and survival after extracellular DNA or dNMPs treatment. The results showed that the extracellular DNA fragments inhibited the growth of D. radiodurans. Interestingly, dGMP, a DNA component, enhanced D. radiodurans tolerance to H(2)O(2) and gamma-radiation significantly. Further experiments indicated that extracellular dGMP stimulated the activity of one catalase (KatA, DR1998), and induced gene transcription including the extracellular nuclease (drb0067). When this only extracellular nuclease gene (drb0067) in D. radiodurans was deleted, the mutant strain showed more sensitive to H(2)O(2) and gamma-radiation than the wild type strain. These results suggest that DRB0067 plays an important role in oxidative stress resistance. Taken together, we proposed a new anti-oxidation mechanism in D. radiodurans. This mechanism acts to increase expression levels of DRB0067 which then secretes active nuclease to degrade extracellular DNA fragments. The extracellular nuclease has a two-fold benefit, creating more free dNTPs for further cell protection and the removal of extracellular DNA fragments.

Function and biochemical characterization of RecJ in Deinococcus radiodurans.

The single-stranded DNA-specific nuclease RecJ is found in most bacteria where it is involved in the RecFOR double-stranded break (DSBs) repair pathway. DSBs repair mainly occurs via the RecFOR pathway in Deinococcus radiodurans, a well-known radiation-resistant bacterium. A recJ null mutant was constructed to investigate the role of recJ in D. radiodurans. recJ inactivation caused growth defects and sensitivity to high temperatures. However, the radiation resistance of the recJ mutant was only moderately decreased. The full-length D. radiodurans RecJ (DrRecJ) protein was expressed and purified to further characterize its biochemical properties. DrRecJ possessed a Mn(2+) concentration-dependent nuclease activity where the optimal Mn(2+) concentration was 0.1mM. DrRecJ had a similar activity profile after adding 10mM Mg(2+) to reactions with different Mn(2+) concentrations, indicating that Mn(2+) is a RecJ regulator. Escherichia coli RecJ has no activity on 5' ssDNA tails shorter than 6-nt, but DrRecJ could effectively degrade DNA with a 4-nt 5' ssDNA tail, suggesting that DrRecJ may have a wider range of DNA substrates. Moreover, SSB in D. radiodurans stimulated the DrRecJ exonuclease activity, whereas DdrB inhibited it and provided protection to ssDNA. Overall, our results indicate that recJ is a nonessential gene in D. radiodurans and that the activity of DrRecJ is regulated by Mn(2+) and SSB-DdrB.

Regulation of MntH by a dual Mn(II)- and Fe(II)-dependent transcriptional repressor (DR2539) in Deinococcus radiodurans.

The high intracellular Mn/Fe ratio observed within the bacteria Deinococcus radiodurans may contribute to its remarkable resistance to environmental stresses. We isolated DR2539, a novel regulator of intracellular Mn/Fe homeostasis in D. radiodurans. Electrophoretic gel mobility shift assays (EMSAs) revealed that DR2539 binds specifically to the promoter of the manganese acquisition transporter (MntH) gene, and that DR0865, the only Fur homologue in D. radiodurans, cannot bind to the promoter of mntH, but it can bind to the promoter of another manganese acquisition transporter, MntABC. ß-galactosidase expression analysis indicated that DR2539 acts as a manganese- and iron-dependent transcriptional repressor. Further sequence alignment analysis revealed that DR2539 has evolved some special characteristics. Site-directed mutagenesis suggested that His98 plays an important role in the activities of DR2539, and further protein-DNA binding activity assays showed that the activity of H98Y mutants decreased dramatically relative to wild type DR2539. Our study suggests that D. radiodurans has evolved a very efficient manganese regulation mechanism that involves its high intracellular Mn/Fe ratio and permits resistance to extreme conditions.