Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells.

BACKGROUND: RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically. METHODS: We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome. RESULTS: We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia. CONCLUSIONS: These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.