The human genome contains regulatory DNA elements, known as enhancers, that can activate gene transcription over long chromosomal distances. Here, we showed that enhancer distance can be critical for gene silencing. We demonstrated that linear recruitment of the normally distal strong HBB enhancer to developmentally silenced embryonic HBE or fetal HBG promoters through deletion or inversion of intervening DNA sequences led to strongly reactivated expression in adult erythroid cells and ex vivo differentiated hematopoietic stem and progenitor cells. A similar observation was made for the HBA locus in which deletion-to-recruit of the distal enhancer strongly reactivated embryonic HBZ expression. Overall, our work assigned function to seemingly nonregulatory genomic segments; by providing linear separation, they may support genes to autonomously control their transcriptional response to distal enhancers.

Chronic lymphocytic leukemia (CLL) is a disease of great clinical and biologic heterogeneity; some patients are observed for years without symptoms, whereas others rapidly develop progressive disease requiring treatment. With therapy, some patients eventually develop resistant CLL or transformation to an aggressive form. Across this spectrum, patients experience immune dysfunction associated with increased risk for infection and second cancers, contributing to morbidity and mortality of the disease. The ultimate therapeutic bull's-eye for CLL is to eliminate the disease and achieve immune restoration. Disease elimination can potentially be achieved for a fraction of patients treated first line with chemoimmunotherapy (fludarabine, cyclophosphamide, and rituximab), for some patients who receive time-limited combined targeted therapy, and for some patients with relapsed/refractory CLL who undergo allogeneic stem cell transplant. Long-term immune restoration for these patients is elusive. Current targeted therapies, including Bruton tyrosine kinase and B-cell lymphoma 2 inhibitors and CD20 monoclonal antibodies used in combinations, can produce exceptional therapeutic outcomes, which are improving survival for patients who need treatment. Although clear progress has been made toward highly effective CLL management, appreciation of the full impact of these advances will require time because of the chronic nature of the disease. In addition, it is imperative to ensure global access to the targeted therapies, emphasizing the need for harmonized regulatory oversight and affordable treatment options worldwide. Here, we discuss research and collaborative strategies to refine the use of targeted agents to eliminate CLL and restore immune function for all affected individuals.

Relapsed/refractory T-cell acute lymphoblastic leukemia (ALL; T-ALL)/lymphoma (LBL) represent a significant unmet medical need. WU-CART-007 is a CD7-targeting, allogeneic, fratricide-resistant chimeric antigen receptor T-cell product generated from healthy donor T cells. WU-CART-007 was evaluated in a phase 1/2 study with a 3+3 dose-escalation design followed by cohort expansion in relapsed/refractory T-ALL/LBL. Patients received 1 infusion of WU-CART-007 after standard or enhanced lymphodepleting chemotherapy. The primary objectives, to characterize safety and assess the composite complete remission rate, were met. Of 28 patients enrolled, 13 received the recommended phase 2 dose (RP2D) of 900 × 106 cells of WU-CART-007 with enhanced lymphodepletion. The most common treatment-related adverse event was cytokine release syndrome (88.5%; 19.2% grade 3-4). Two grade 1 immune effector cell-associated neurotoxicity syndrome events (7.7%) and 1 grade 2 acute graft-versus-host disease event occurred (3.8%). One grade 2 immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome was observed. Among the 11 patients evaluable for response at the RP2D who received enhanced lymphodepleting chemotherapy, the overall response rate was 90.9%, and the composite complete remission rate was 72.7%. WU-CART-007 at the RP2D demonstrated a high response rate in patients with relapsed/refractory T-ALL/LBL and has the potential to provide a new treatment option. This trial was registered at www.ClinicalTrials.gov as #NCT04984356.

The integrity of the hematopoietic stem cell (HSC) pool depends on effective long-term self-renewal and the timely elimination of damaged or differentiation-prone HSCs. Although the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response has been shown to initiate proapoptotic signaling in response to endoplasmic reticulum (ER) stress in vitro, its role in regulating the HSC fate in vivo remains incompletely understood. Here, we demonstrated that PERK is dispensable for steady-state hematopoiesis and HSC self-renewal under homeostatic conditions. However, under ER stress induced by the disruption of ER-associated degradation (ERAD) through knockout of key components, such as Sel1L or Hrd1, PERK becomes activated and drives HSC proliferation and depletion. Notably, deletion of PERK or expression of a kinase-dead PERK mutant significantly rescued the HSC defects caused by Sel1L or Hrd1 loss. Mechanistically, an ERAD deficiency did not lead to increased HSC apoptosis or elevated reactive oxygen species, and PERK knockout had minimal impact on HSC apoptosis. Instead, PERK activation promoted aberrant mTOR (mammalian target of rapamycin) signaling and HSC hyperproliferation, ultimately compromising self-renewal capacity. This PERK-driven elimination of stressed HSCs may function as a protective mechanism to maintain the overall HSC pool integrity. Collectively, our findings reveal a previously unrecognized, proliferative, and apoptosis-independent role for PERK in regulating HSC fate under ER stress, highlighting a novel mechanism for preserving HSC homeostasis.

Inotuzumab ozogamicin (InO) is an antibody-calicheamicin conjugate with high efficacy in lymphoid malignancies. It targets the B-cell surface protein CD22, which is expressed in most B-cell acute lymphoblastic leukemia (B-ALL) cases, albeit with variable intensity. However, factors governing CD22 expression and thus leukemia sensitivity to InO remain incompletely understood. Using multiomic characterization of 196 human B-ALL samples, coupled with ex vivo InO sensitivity profiling, we showed that early leukemia differentiation arrest at the pre-pro-B stage is associated with resistance to InO. Screening of 1639 transcription factor genes identified early B-cell factor 1 (EBF1) as a key regulator of CD22 expression (false discovery rate of 7.1 × 10-4). When comparing the assay for transposase-accessible chromatin with sequencing profiling results of the most InO-sensitive and -resistant cases (50% lethal concentration <10th vs >90th percentile, n = 18), the binding motif for EBF1 was strikingly enriched in regions with differential open chromatin status (P = 8 × 10-174). CRISPR interference targeting EBF1 binding sites at the CD22 locus led to an ∼50-fold reduction in cell surface CD22 expression and, consequently, an ∼22-fold increase in InO resistance in ALL cell lines. Interestingly, within BCR::ABL1 ALL, we observed intrasubtype heterogeneity linked to EBF1 transcriptional downregulation (P = 1.1 × 10-15) and/or somatic alteration (P = .004), which led to reduced CD22 expression (P = 8.3 × 10-11) and ex vivo and in vivo resistance to InO. Collectively, these findings point to the direct impact of EBF1 on CD22 expression during B-cell development, which, in turn, contributes to interpatient variability in InO response, even within the same subtype of B-ALL.