Human pluripotent stem cells (hPSCs) can be used to investigate hematopoietic development and have the potential to advance cell-based therapies and to facilitate developmental biology studies. However, efficient ex vivo differentiation into hematopoietic lineages, including red blood cells (RBCs) of the erythroid lineage and immune cells such as macrophages of the myeloid lineage, is hampered by the need for precise temporal regulation of cytokines and growth factors. In this study, we developed an optimized protocol for hematopoietic lineage specification from hPSCs by fine-tuning the temporal dynamics of cytokine and growth factor applications. Prolonged mesodermal specification in the absence of hemogenic cytokines significantly enhanced the generation of hematopoietic progenitors (CD34＋CD45＋) with robust functional potential. Early administration of interleukin (IL)-3 during hematopoietic specification promoted progenitor expansion and maturation. Supplementation of bone morphogenetic protein 4 at the hematopoietic maturation stage enhanced the differentiation efficiency and preferentially drove myeloid lineage commitment toward macrophages at the expense of erythroid differentiation. The timing of erythropoietin administration was important in erythroid lineage commitment, and delayed treatment (day 10) enhanced erythroblast expansion and RBC production. By contrast, the timing of IL-6, GM-CSF, and M-CSF exposure did not significantly affect macrophage differentiation efficiency, suggesting that myeloid lineage specification follows a default pathway under optimized differentiation conditions. These findings suggest a refined, time-controlled strategy for directing hematopoietic differentiation from hPSCs, and provide insight into therapeutic blood cell production, regenerative medicine, and ex vivo modeling of hematopoietic disorders.

Genetic mutations are being thoroughly mapped in human cancers, yet a fundamental question in cancer biology is whether such mutations are functionally required for cancer initiation, maintenance of established cancer, or both. Here, we study this question in the context of human acute myeloid leukemia (AML), where DNMT3AR882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the DNA methylation landscape to initiate leukemia. We developed CRISPR-based methods to directly correct DNMT3AR882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3AR882 mutations were largely dispensable for disease maintenance. Replacing DNMT3AR882 mutants with wild-type DNMT3A did not impair the ability of AML cells to engraft in vivo, and minimally altered DNA methylation. Taken together, DNMT3AR882 mutations are initially necessary for AML initiation, but are largely dispensable for disease maintenance. The notion that initiating oncogenes differ from those that maintain cancer has important implications for cancer evolution and therapy.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in treating hematologic cancers, but resistance is common and efficacy is limited in solid tumors. We found that CAR T cells autonomously propagate epigenetically programmed type I interferon signaling through chronic stimulation, which hampers antitumor function. EGR2 transcriptional regulator knockout not only blocks this type I interferon-mediated inhibitory program but also independently expands early memory CAR T cells with improved efficacy against liquid and solid tumors. The protective effect of EGR2 deletion in CAR T cells against chronic antigen-induced exhaustion can be overridden by interferon-β exposure, suggesting that EGR2 ablation suppresses dysfunction by inhibiting type I interferon signaling. Finally, a refined EGR2 gene signature is a biomarker for type I interferon-associated CAR T cell failure and shorter patient survival. These findings connect prolonged CAR T cell activation with deleterious immunoinflammatory signaling and point to an EGR2-type I interferon axis as a therapeutically amenable biological system.
To improve CAR T cell therapy outcomes, modulating molecular determinants of CAR T cell-intrinsic resistance is crucial. Editing the gene encoding the EGR2 transcriptional regulator renders CAR T cells impervious to type I interferon pathway-induced dysfunction and improves memory differentiation, thereby addressing major barriers to progress for this emerging class of cancer immunotherapies. This article is highlighted in the In This Issue feature, p. 1501.
©2023 American Association for Cancer Research.

Background: Patients with head and neck squamous cell carcinoma (HNSCC) are at high risk of developing locoregional recurrence and secondary cancers. Early prediction is crucial for improving outcomes. This study evaluates the prognostic and surveillance utilities of circulating tumour cells (CTCs) in post-treatment HNSCC patients. Methods Blood samples were collected from 154 HNSCC patients at baseline and follow-up time points and CTC was isolated with a microfluid device. Recurrence and death due to cancer were assessed during the follow-up period. Results In patients with HNSCC, the presence of CTCs at baseline was an independent predictor of recurrence (odds ratio = 1.55, p < 0.05) and death (odds ratio = 2.10, p < 0.01), even after adjusting for TNM or nodal stage. Patients with CTC at baseline experienced poorer survival outcomes (p < 0.0001). Additionally, our study found that patients with CTCs in a follow-up appointment were 2.5 times more likely to experience recurrence or death from HNSCC (p < 0.05) prior to their next clinical visit. Conclusions Our study highlights CTCs' potential as a prognostic marker for risk stratification in HNSCC patients. Early CTC detection enables precise risk assessment, guiding treatment adjustments and ultimately improving patient outcomes.

Tumor-infiltrating lymphocytes (TILs), especially CD8+ TILs, represent a favorable prognostic factor in high-grade serous ovarian cancer (HGSOC) and other tumor lineages. Here, we analyze the spatial heterogeneity of different TIL subtypes in HGSOC. We integrated RNA sequencing, whole-genome sequencing, bulk T cell receptor (TCR) sequencing, as well as single-cell RNA/TCR sequencing to investigate the characteristics and differential composition of TILs across different HGSOC sites. Two immune "cold" patterns in ovarian cancer are identified: (1) ovarian lesions with low infiltration of mainly dysfunctional T cells and immunosuppressive Treg cells and (2) omental lesions infiltrated with non-tumor-specific bystander cells. Exhausted CD8 T cells that are preferentially enriched in ovarian tumors exhibit evidence for expansion and cytotoxic activity. Inherent tumor immune microenvironment characteristics appear to be the main contributor to the spatial differences in TIL status. The landscape of spatial heterogeneity of TILs may inform potential strategies for therapeutic manipulation in HGSOC.
Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.