Allogeneic transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5-null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs engraft and reconstitute multiple hematopoietic lineages in vivo and can be engineered to express multiple antibodies simultaneously (in pre-clinical models). Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the foundation for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.
© 2025. The Author(s).

Our study explores the complex dynamics of the integrated stress response (ISR) axis, highlighting PIM2 kinase's critical role and its interaction with the BCL2 protein family, uncovering key mechanisms of cell survival and tumor progression. Elevated PIM2 expression, a marker of various cancers, often correlates with disease aggressiveness. Using a model of normal and malignant plasma cells, we show that inhibiting PIM2 kinase inhibits phosphorylated BAD production and activates ISR-mediated NOXA expression. This shift towards MCL1 dependence underscores the synergy achieved through combined PIM/MCL1 inhibition, driven largely by ISR-mediated NOXA expression. In mouse xenograft models, dual targeting of PIM2 and MCL1 effectively controls tumor growth-a response reversed by ISR-specific inhibition and upregulation of genes linked to tumor cell dissemination. This work elucidates the molecular intricacies of PIM2 inhibition and its implications for cancer therapy, especially in tumors with elevated PIM2 expression.
© 2024. The Author(s).

To fully utilize the potential of CRISPR-Cas9-mediated genome editing, time-restricted and targeted delivery is crucial. By modulating the pseudotype of engineered lentivirus-derived nanoparticles (LVNPs), we demonstrate efficient cell-targeted delivery of Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes, supporting gene modification in a defined subset of cells in mixed cell populations. LVNPs pseudotyped with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein resulted in angiotensin-converting enzyme 2 (ACE2)-dependent insertion or deletion (indel) formation in an ACE2+/ACE2- population of cells, whereas Nipah virus glycoprotein pseudotyping resulted in Ephrin-B2/B3-specific gene knockout. Additionally, LVNPs pseudotyped with Edmonston strain measles virus glycoproteins (MV-H/F) delivered Cas9/sgRNA RNPs to CD46+ cells with and without additional expression of SLAM (signaling lymphocytic activation molecule; CD150). However, an engineered SLAM-specific measles virus pseudotype (measles virus-hemagglutinin/fusion [MV-H/F]-SLAM) efficiently targeted LVNPs to SLAM+ cells. Lentiviral vectors (LVs) pseudotyped with MV-H/F-SLAM efficiently transduced >80% of interleukin (IL)-4/IL-21-stimulated primary B cells cultured on CD40 ligand (CD40L)-expressing feeder cells. Notably, LVNPs pseudotyped with MV-H/F and MV-H/F-SLAM reached indel rates of >80% and >60% in stimulated primary B cells, respectively. Collectively, our findings demonstrate the modularity of LVNP-directed delivery of ready-to-function Cas9/sgRNA complexes. Using a panel of different pseudotypes, we provide evidence that LVNPs can be engineered to induce effective indel formation in a subpopulation of cells defined by the expression of surface receptors.
© 2024 The Author(s).

Ligation of the B cell antigen receptor (BCR) initiates humoral immunity. However, BCR signaling without appropriate co-stimulation commits B cells to death rather than to differentiation into immune effector cells. How BCR activation depletes potentially autoreactive B cells while simultaneously primes for receiving rescue and differentiation signals from cognate T lymphocytes remains unknown. Here, we use a mass spectrometry-based proteomic approach to identify cytosolic/nuclear shuttling elements and uncover transcription factor EB (TFEB) as a central BCR-controlled rheostat that drives activation-induced apoptosis, and concurrently promotes the reception of co-stimulatory rescue signals by supporting B cell migration and antigen presentation. CD40 co-stimulation prevents TFEB-driven cell death, while enhancing and prolonging TFEB's nuclear residency, which hallmarks antigenic experience also of memory B cells. In mice, TFEB shapes the transcriptional landscape of germinal center B cells. Within the germinal center, TFEB facilitates the dark zone entry of light-zone-residing centrocytes through regulation of chemokine receptors and, by balancing the expression of Bcl-2/BH3-only family members, integrates antigen-induced apoptosis with T cell-provided CD40 survival signals. Thus, TFEB reprograms antigen-primed germinal center B cells for cell fate decisions.
© 2024. The Author(s).

The use of Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib achieves a remarkable clinical response in mantle cell lymphoma (MCL). Acquired drug resistance, however, is significant and affects long-term survival of MCL patients. Here, we demonstrate that DNA methyltransferase 3A (DNMT3A) is involved in ibrutinib resistance. We find that DNMT3A expression is upregulated upon ibrutinib treatment in ibrutinib-resistant MCL cells. Genetic and pharmacological analyses reveal that DNMT3A mediates ibrutinib resistance independent of its DNA-methylation function. Mechanistically, DNMT3A induces the expression of MYC target genes through interaction with the transcription factors MEF2B and MYC, thus mediating metabolic reprogramming to oxidative phosphorylation (OXPHOS). Targeting DNMT3A with low-dose decitabine inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting DNMT3A-mediated metabolic reprogramming to OXPHOS with decitabine provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.