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Morroniside is known to improve osteoporosis by promoting osteoblastogenesis. The activation of PI3K/Akt/mTOR signaling is a significant mechanism in morroniside-promoted osteoblastogenesis. It is well known that protective autophagy is an important factor in osteoblastogenesis. However, the activation of mTOR signaling can inhibit autophagy. This study aimed to investigate the relationship between mTOR signaling and autophagy in morroniside-regulated osteoblastogenesis. In this study, we investigated the effect of morroniside on the autophagic activity (LC3 conversion rate, LC3-puncta formation, and autophagosome number) of differentiated osteoblast precursors (MC3T3-E1 cells). Then, we identified the roles of mTOR knockdown in morroniside-regulated alterations of autophagy and osteogenic parameters in MC3T3-E1 cells. Next, mTOR knockdown and overexpression were used to observe the roles of mTOR in morroniside-regulated alterations of autophagic molecules (Atg7, Atg13, and Beclin1). Subsequently, the additional value of the above autophagic molecules on morroniside-regulated osteogenic parameters in MC3T3-E1 cells was analyzed based on lentiviral transduction. Finally, combined with morroniside and TAT-Beclin1, the roles of Beclin1 upregulation in the in vivo effects of morroniside was investigated. Our experimental data showed that morroniside promoted both the mTOR activity and autophagy in MC3T3-E1 cells. Morroniside-upregulated autophagic activity and Atg13 or Beclin1 protein level in MC3T3-E1 cells were enhanced by mTOR knockdown. Furthermore, Morroniside-upregulated Atg13 and Beclin1 expression was reversed by mTOR overexpression. Importantly, autophagy upregulation with overexpression of the autophagic gene, Atg13 or BECN1 (gene form of Beclin1), significantly promoted osteoblastogenesis regulated by morroniside. The promotional effect of morroniside on bone microarchitecture, bone mass, and bone parameters (including trabecular bone area and OCN expression in trabecular bone) in ovariectomized (OVX) mice was enhanced by TAT-Beclin1 administration. In conclusion, the autophagy-enhancing drugs related to Beclin1 or Atg13 may be an effective adjuvant therapy in the treatment of osteoporosis with morroniside.

A new layer of transcriptional control N 6 -methyladenosine (m 6 A) is the most abundant messenger RNA modification in almost all eukaryotes. Liu et al. now show that m 6 A is also cotranscriptionally added onto various chromosome-associated regulatory RNAs (carRNAs) in mammalian cells. Disruption of m 6 A modification of these RNAs increases their abundance and promotes gene transcription by increasing the chromatin accessibility. Thus, m 6 A serves as a switch to regulate carRNA levels by tuning nearby chromatin state and downstream transcription. Science , this issue p. 580

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.

Objective: To determine the efficacy of acupuncture around the greater tuberosity of the femur (AGTF) combined with acupuncture at Xuehai acupoint for postoperative pain in elderly patients with intertrochanteric fracture. Methods: A total of 97 elderly patients with intertrochanteric fracture treated by proximal femoral nail antirotation (PFNA) were enrolled and randomly assigned into an observation group (Obs group, n=48) and a control group (Con group, n=49). The Obs group was treated by aspirin and AGTF combined with acupuncture at Xuehai acupoint for analgesia, while the Con group was treated by aspirin alone for analgesia. Both groups were treated for 7 consecutive days. The two groups were compared in pain degree (visual analog scale (VAS) score) after operation and hip joint function (Harris score), daily living ability (modified Barthel index (MBI) score), bone metabolism-related indexes, and inflammatory factors before and after treatment. Results: At 1-7 d after operation, both groups had gradually lower VAS scores, and at 5 and 7 d after operation, the Obs group had a lower VAS score than the Con group (both P0.05). Moreover, at 7 d after operation, both groups showed a decrease in C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α), and the two levels in the Obs group were lower than those in the Con group (both P

Tumor-associated macrophages (TAMs) can dampen the antitumor activity of T cells, yet the underlying mechanism remains incompletely understood. Here, we show that C1q

There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies1,2. Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response3,4. Here we show that durable neoantigen-specific immunity is regulated by mRNA N6-methyadenosine (m6A) methylation through the m6A-binding protein YTHDF15. In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8+ T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8+ T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by m6A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1-/- mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy.

Introduction Acute myeloid leukemia (AML) is an aggressive disease with genetic and phenotypic heterogeneity that results in a highly variable response to standard chemotherapy. Azacitidine (AZA) is a hypomethylating agent (HMA) and has been investigated in combination with intensive chemotherapy as an epigenetic primer to sensitize leukemic cells to treatment. In a phase 1 trial, this regimen was safe and well-tolerated with overall response rate (CR+CRi) of 61% and complete remission rate of 41% (Cahill et al, Blood Adv 2020). Predictive biomarkers for response to this treatment strategy have not yet been identified. Since 5-hydroxymethylcytosine (5hmC) is an epigenetic biomarker in cancer, we hypothesized that Nano-5hmC-Seal sequencing technology may serve as a novel approach to identifying 5hmC profiles predictive of treatment response to epigenetic priming. Methods We performed RNA-seq gene expression and Nano-5hmC-Seal DNA profiling from peripheral blood/bone marrow samples of patients with high-risk AML to identify potential 5hmC profile biomarkers and gene expression changes (Figure 1A). Patients (n=46) were treated in a 3+3 dose-escalation scheme of AZA (37.5 mg/m 2, 50 mg/m 2, or 75 mg/m 2) on days 1-5 followed by high-dose cytarabine (3000 mg/m 2) and mitoxantrone (30 mg/m 2) (AZA-HiDAC-Mito) on day 6 and day 10 in a phase 1 trial previously reported (Cahill et al, Blood Adv 2020). We compared pre-treatment RNA-seq gene expression and 5hmC DNA profiles between responders (CR+CRi) and non-responders, as well as between pre-treatment and after 5 days of AZA for individual patients. We used an XGBoost machine learning model in Python based on a training set of patients to develop a 5hmC gene signature to predict response to AZA-HiDAC-Mito in an independent test set of patients. We compared continuous variables with two-tailed Student's t-test and used the Kaplan-Meier method with log-rank test for survival analysis. Results Thirty-three patients (72%) had adequate RNA samples for RNA-seq gene expression analysis. Eighteen responded to treatment (CR +CRi) and were enriched with gene expression patterns involved in cell-cell interaction and activation of cell cycle, while non-responders (n=15) had a higher expression of leukemic stem cell (LSC) signatures. There was no difference in gene expression profile when comparing pre-treatment samples to day 5 samples after AZA exposure. From the 5hmC profiling [n=40 (87%) patients with adequate samples], increased 5hmC in LSC genes was associated with treatment resistance to AZA-HiDAC-Mito (p=0.044). The number of differentially hydroxy-methylated genes (DhMGs) increased with higher doses of AZA exposure suggesting a dose-dependent epigenetic effect from AZA. Patients with a greater number of DhMGs following 5 days of AZA treatment had improved survival (p=0.015) (Figure 1B). Using the 5hmC-based XGBoost machine learning model comparing 5hmC profiles between responders to non-responders from a training set of patients (n=22), we developed an 11-gene 5hmC pre-treatment signature (including SKP1, WNT8A, CYP2E1, and NBPF9) to predict treatment response. The model was highly effective in predicting response to therapy, with an area under the curve (AUC) of 0.86 in an independent test set of patients (n=18) treated with AZA-HiDAC-Mito (Figure 1C). Conclusion In patients with AML treated with AZA-HiDAC-Mito, a pre-treatment LSC gene expression signature enriched with 5hmC was associated with treatment resistance. More DhMGs at day 5 appear to be a dose-dependent epigenetic effect that is induced by AZA and is associated with longer survival despite the absence of an immediate change in gene expression levels. An 11-gene 5hmC pre-treatment signature may be a predictive biomarker for AZA-HiDAC-Mito therapy and other HMA-based approaches. These findings warrant validation in a larger prospective trial. Figure 1 Figure 1. Disclosures Zhang: Bristol-Myers Squibb: Current Employment. Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Odenike: Celgene, Incyte, AstraZeneca, Astex, NS Pharma, AbbVie, Gilead, Janssen, Oncotherapy, Agios, CTI/Baxalta, Aprea: Research Funding; AbbVie, Celgene, Impact Biomedicines, Novartis, Taiho Oncology, Takeda: Consultancy. He: Epican Genetech: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Summary Tumor-associated macrophages (TAMs) can dampen the antitumor activity of T cells, yet the underlying mechanism remains incompletely understood. Here, we show that C1q+ TAMs are regulated by an RNA N6-methyladenosine (m6A) program and modulate tumor-infiltrating CD8+ T cells by expressing multiple immunomodulatory ligands. Macrophage-specific knockout of an m6A methyltransferase Mettl14 drives CD8+ T cell differentiation along a dysfunctional trajectory, impairing CD8+ T cells to eliminate tumors. Mettl14-deficient C1q+ TAMs show a decreased m6A abundance on and a higher level of transcripts of Ebi3, a cytokine subunit. In addition, neutralization of EBI3 leads to reinvigoration of dysfunctional CD8+ T cells and overcomes immunosuppressive impact in mice. We show that the METTL14-m6A levels are negatively correlated with dysfunctional T cell levels in patients with colorectal cancer, supporting the clinical relevance of this regulatory pathway. Thus, our study demonstrates how an m6A methyltransferase in TAMs promotes CD8+ T cell dysfunction and tumor progression.

Table S2. Sequences of targeting crRNAs used for in vitro RNA transcription. Table S3. Target sequences harboring various 5â ˛ PAM sequences used for in vitro DNA cleavage assay. Table S4. Protospacer sequences used for genome editing. Table S5. crRNA scaffold optimization and screening. Table S6. Primer sequences used for PCR amplification. Table S7. Frequency of Cas12a- and SpCas9-mediated targeted indel mutations at on-target sites in human 293FT cells. Table S8. Off-target analysis by targeted deep sequencing in the human 293FT cells. Table S9. Off-target analysis by whole genome sequencing (WGS) in human 293FT cells. (PDF 395 kb)

Supplementary Sequences. The humanized Cas12a coding sequences, protein sequences, U6-crRNA backbone sequences, mammalian and prokaryotic expression vector sequences used in this study. (PDF 183 kb)

Supplementary methods. (PDF 124 kb)

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.

In this Letter, a citation to ‘Fig. 1e’ has been corrected to ‘Fig. 1d’ in the sentence starting “By contrast, the anti-tumour response…”. This has been corrected online