CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.

Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6 A and m5 C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq). Using CIGAR-seq, we discovered NSUN6 as a novel mRNA m5 C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non-overlapping subsets of mRNA m5 C sites and together contributed to almost all the m5 C modification in mRNA. Finally, using m1 A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification.

ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerization.

Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed "ActuAtor" that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.

Single cell multi-omics reveal intra-cell-line heterogeneity across human cancer cell lines.

Human cancer cell lines have long served as tools for cancer research and drug discovery, but the presence and the source of intra-cell-line heterogeneity remain elusive. Here, we perform single-cell RNA-sequencing and ATAC-sequencing on 42 and 39 human cell lines, respectively, to illustrate both transcriptomic and epigenetic heterogeneity within individual cell lines. Our data reveal that transcriptomic heterogeneity is frequently observed in cancer cell lines of different tissue origins, often driven by multiple common transcriptional programs. Copy number variation, as well as epigenetic variation and extrachromosomal DNA distribution all contribute to the detected intra-cell-line heterogeneity. Using hypoxia treatment as an example, we demonstrate that transcriptomic heterogeneity could be reshaped by environmental stress. Overall, our study performs single-cell multi-omics of commonly used human cancer cell lines and offers mechanistic insights into the intra-cell-line heterogeneity and its dynamics, which would serve as an important resource for future cancer cell line-based studies.

Sotrovimab restores neutralization against current Omicron subvariants in patients with blood cancer.

Wu et al. report that patients with hematologic malignancies have reduced immunity against SARS-CoV-2 Omicron subvariants and Sotrovimab retains neutralizing capacity against all tested Omicron subvariants.

Functional immune responses against SARS-CoV-2 variants of concern after fourth COVID-19 vaccine dose or infection in patients with blood cancer.

Patients with blood cancer continue to have a greater risk of inadequate immune responses following three COVID-19 vaccine doses and risk of severe COVID-19 disease. In the context of the CAPTURE study (NCT03226886), we report immune responses in 80 patients with blood cancer who received a fourth dose of BNT162b2. We measured neutralizing antibody titers (NAbTs) using a live virus microneutralization assay against wild-type (WT), Delta, and Omicron BA.1 and BA.2 and T cell responses against WT and Omicron BA.1 using an activation-induced marker (AIM) assay. The proportion of patients with detectable NAb titers and T cell responses after the fourth vaccine dose increased compared with that after the third vaccine dose. Patients who received B cell-depleting therapies within the 12 months before vaccination have the greatest risk of not having detectable NAbT. In addition, we report immune responses in 57 patients with breakthrough infections after vaccination.

Adaptive immunity and neutralizing antibodies against SARS-CoV-2 variants of concern following vaccination in patients with cancer: the CAPTURE study.

Coronavirus disease 2019 (COVID-19) antiviral response in a pan-tumor immune monitoring (CAPTURE) ( NCT03226886 ) is a prospective cohort study of COVID-19 immunity in patients with cancer. Here we evaluated 585 patients following administration of two doses of BNT162b2 or AZD1222 vaccines, administered 12 weeks apart. Seroconversion rates after two doses were 85% and 59% in patients with solid and hematological malignancies, respectively. A lower proportion of patients had detectable titers of neutralizing antibodies (NAbT) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) versus wild-type (WT) SARS-CoV-2. Patients with hematological malignancies were more likely to have undetectable NAbT and had lower median NAbT than those with solid cancers against both SARS-CoV-2 WT and VOC. By comparison with individuals without cancer, patients with hematological, but not solid, malignancies had reduced neutralizing antibody (NAb) responses. Seroconversion showed poor concordance with NAbT against VOC. Previous SARS-CoV-2 infection boosted the NAb response including against VOC, and anti-CD20 treatment was associated with undetectable NAbT. Vaccine-induced T cell responses were detected in 80% of patients and were comparable between vaccines or cancer types. Our results have implications for the management of patients with cancer during the ongoing COVID-19 pandemic.

Adaptive immunity and neutralizing antibodies against SARS-CoV-2 variants of concern following vaccination in patients with cancer: The CAPTURE study.

CAPTURE (NCT03226886) is a prospective cohort study of COVID-19 immunity in patients with cancer. Here we evaluated 585 patients following administration of two doses of BNT162b2 or AZD1222 vaccines, administered 12 weeks apart. Seroconversion rates after two doses were 85% and 59% in patients with solid and hematological malignancies, respectively. A lower proportion of patients had detectable neutralizing antibody titers (NAbT) against SARS-CoV-2 variants of concern (VOCs) vs wildtype (WT). Patients with hematological malignancies were more likely to have undetectable NAbT and had lower median NAbT vs solid cancers against both WT and VOCs. In comparison with individuals without cancer, patients with haematological, but not solid, malignancies had reduced NAb responses. Seroconversion showed poor concordance with NAbT against VOCs. Prior SARS-CoV-2 infection boosted NAb response including against VOCs, and anti-CD20 treatment was associated with undetectable NAbT. Vaccine-induced T-cell responses were detected in 80% of patients, and were comparable between vaccines or cancer types. Our results have implications for the management of cancer patients during the ongoing COVID-19 pandemic.

Integrative multi-omics analysis of a colon cancer cell line with heterogeneous Wnt activity revealed RUNX2 as an epigenetic regulator of EMT.

Epithelial-mesenchymal transition (EMT) program, which facilitates tumor metastasis, stemness and therapy resistance, is a reversible biological process that is largely orchestrated at the epigenetic level under the regulation of different cell signaling pathways. EMT state is often heterogeneous within individual tumors, though the epigenetic drivers underlying such heterogeneity remain elusive. In colon cancer, hyperactivation of the Wnt/ß-catenin signaling not only drives tumor initiation, but also promotes metastasis in late stage by promoting EMT program. However, it is unknown whether the intratumorally heterogeneous Wnt activity could directly drive EMT heterogeneity, and, if so, what are the underlying epigenetic driver(s). Here, by analyzing a phenotypically and molecularly heterogeneous colon cancer cell line using single-cell RNA sequencing, we identified two distinct cell populations with positively correlated Wnt activity and EMT state. Integrative multi-omics analysis of these two cell populations revealed RUNX2 as a critical transcription factor epigenetically driving the EMT heterogeneity. Both in vitro and in vivo genetic perturbation assays validated the EMT-enhancing effect of RUNX2, which remodeled chromatin landscape and activated a panel of EMT-associated genes through binding to their promoters and/or potential enhancers. Finally, by exploring the clinical data, we showed that RUNX2 expression is positively correlated with metastasis development and poor survival of colon cancer patients, as well as patients afflicted with other types of cancer. Taken together, our work revealed RUNX2 as a new EMT-promoting epigenetic regulator in colon cancer, which may potentially serve as a prognostic marker for tumor metastasis.