Yamin Chen

B lymphocytes are essential mediators of humoral immunity and play multiple roles in human cancer. To decode the functions of tumor-infiltrating B cells, we generated a B cell blueprint encompassing single-cell transcriptome, B cell-receptor repertoire, and chromatin accessibility data across 20 different cancer types (477 samples, 269 patients). B cells harbored extraordinary heterogeneity and comprised 15 subsets, which could be grouped into two independent developmental paths (extrafollicular versus germinal center). Tumor types grouped into the extrafollicular pathway were linked with worse clinical outcomes and resistance to immunotherapy. The dysfunctional extrafollicular program was associated with glutamine-derived metabolites through epigenetic-metabolic cross-talk, which promoted a T cell-driven immunosuppressive program. These data suggest an intratumor B cell balance between extrafollicular and germinal-center responses and suggest that humoral immunity could possibly be harnessed for B cell-targeting immunotherapy.

BACKGROUND: Mirk/Dyrk1B contributes to G0 arrest by destabilization of cyclin D1 and stabilization of p27kip1 to maintain the viability of quiescent human cancer cells, and it could be negatively regulated by mitogenic-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling. This study was performed to investigate the effect of Mirk/Dyrk1B on cell cycle and survival of human cancer cells involving MAPK/ERK signaling. METHODS: The correlations between Mirk/Dyrk1B expression and active ERK1/2 detected by western blot in both ovarian cancer and non-small cell lung cancer (NSCLC) cells were analyzed by simple regression. Mirk/Dyrk1B unique phosphopeptides with sites associated with Mirk/Dyrk1B protein were isolated and quantitated by liquid chromatography coupled to tandem mass/mass spectrometry (LC-MS/MS) proteomics analysis. The human cancer cells were treated with small interfering RNAs (siRNAs) and/or U0126, an inhibitor of MEK for indicated duration, followed by investigating the alterations of cell cycle and apoptosis as well as related proteins examined by flow cytometry and Western blot, respectively. RESULTS: Our study demonstrated the widely expressed Mirk/Dyrk1B proteins in the human cancer cells were positively correlated with the levels of activated ERK1/2. Moreover, Mirk/Dyrk1B protein expressions consistent with the tyrosine autophosphorylated levels in the human cancer cells were increased by U0126 or growth factor-depleted culture. Conversely, knockdown of Mirk/Dyrk1B by siRNA led to up-regulated activation of c-Raf-MEK-ERK1/2 pathway and subsequent changes in cell cycle proteins (cyclin D1, p27kip1), accompanied by increased growth rate and cells from G0/G1 into S of cell cycle which could be blocked by U0126 in a dose-dependent manner, indicating Mirk/Dyrk1B may sequester MAPK/ERK pathway, and vice versa. Whereas, combined Mirk siRNA and U0126 induced cell apoptosis in the human cancer cells. CONCLUSIONS: These data together show that Mirk/Dyrk1B mediates cell cycle and survival via interacting with MAPK/ERK signals and simultaneous inhibition of both pathways may be a novel therapeutic target for human cancer.

Neutralizing antibodies (nAbs) play an important role against SARS-CoV-2 infections. Previously, we have reported one potent receptor binding domain (RBD)-binding nAb Ab08 against the SARS-CoV-2 prototype and a panel of variants, but Ab08 showed much less efficacy against the variants harboring the L452R mutation. To overcome the antibody escape caused by the L452R mutation, we generated several structure-based Ab08 derivatives. One derivative, Ab08-K99E, displayed the mostly enhanced neutralizing potency against the Delta pseudovirus bearing the L452R mutation compared to the Ab08 and other derivatives. Ab08-K99E also showed improved neutralizing effects against the prototype, Omicron BA.1, and Omicron BA.4/5 pseudoviruses. In addition, compared to the original Ab08, Ab08-K99E exhibited high binding properties and affinities to the RBDs of the prototype, Delta, and Omicron BA.4/5 variants. Altogether, our findings report an optimized nAb, Ab08-K99E, against SARS-CoV-2 variants and demonstrate structure-based optimization as an effective way for antibody development against pathogens.

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5′-NRN-3′, 5′-NTA-3′, and 5′-NCK-3′ could be considered as canonical PAMs. 5′-NCA-3′ and 5′-NTK-3′ may serve as non-priority PAMs. At the same time, PAM of 5′-NYC-3′ is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.