Cieniewicz, B. | Search bioRxiv

bioRxiv2023

Therapeutic Targeting of TIM-4-L With Engineered T Cells for Acute Myeloid Leukemia

Disruption of the lipid asymmetric bilayer is a common feature observed in cancer cells. We utilized the natural immune receptor TIM-4 to interrogate for loss of plasma membrane phospholipid polarity in primary acute myelogenous leukemia (AML) samples. We performed FACs analysis in 33 patients and correlated TIM-4-L expression frequency and intensity with molecular disease characteristics. In normal tissues, TIM-4-L is confined to the internal leaflet of the plasma membrane. By contrast, 86% of untreated AML blasts in our analysis displayed upregulation of cell surface TIM-4-L. These observations were agnostic to AML genetic classification, as samples with mutations in TP53, ASXL1, and RUNX1, also displayed TIM-4-L upregulation similar to that seen in favorable and intermediate subtypes. This TIM-4-L dysregulation was also stably present in both Kasumi-1 and MV-4-11 AML cell lines. To evaluate the potential of upregulated TIM-4-L to serve as a target for adoptive T cell therapy (ACT), we constructed TIM-4-L-directed engineered T cells, which demonstrated potent anti-leukemic effects, effectively eliminating AML cell lines both in vitro and in vivo. This approach led to the eradication of AML cells across a range of endogenous TIM-4-L expression levels. These results highlight TIM-4-L as a highly prevalent and novel target for T cell-based therapy in AML. Further investigations into the role of TIM-4-L in AML pathogenesis and its potential as an anti-leukemic target for clinical development are warranted.

cancer biology↗

bioRxiv2022

IKKalpha-Mediated Non-canonical NF-kappaB Signaling is Required to Support Murine Gammaherpesvirus 68 Latency In Vivo

Non-canonical NF-kappaB signaling is activated in B cells via TNF receptor superfamily members CD40, Lymphotoxin beta-R, and BAFF-R. The non-canonical pathway is required at multiple stages of B-cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt non-canonical NF-kappaB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IKKalpha, named IKK-SA, with S176A and S180A mutations that prevent phosphorylation by NIK. We controlled for the transgene insertion by introducing two all-frame stop codons into the IKK-SA gene. The IKK-SA mutant but not the IKK-SA.STOP control virus impaired LTbetaR-mediated activation of NF-kappaB p52 upon fibroblast infection. IKK-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKK-SA mutant was severely defective in colonization of the spleen and in the establishment of latency compared to the IKK-SA.STOP control and WT MHV68 at 16 dpi. Reactivation was undetectable in splenocytes infected with the IKK-SA mutant, but reactivation in peritoneal cells was not impacted by IKK-SA. Taken together, the non-canonical NF-kappaB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68. IMPORTANCEThe latency programs of the human gammaherpesviruses EBV and KSHV are associated with B cell lymphomas. It is critical to understand the signaling pathways that are used by gammaherpesviruses to establish and maintain latency in primary B cells. We used a novel approach to block non-canonical NF-kappaB signaling only in the infected cells of mice. We generated a recombinant virus that expresses a dominant negative mutant of IKKalpha that is non-responsive to upstream activation. Latency was reduced in a route- and cell type-dependent manner in mice infected with this recombinant virus. These findings identify a significant role for the non-canonical NF-kappaB signaling pathway that might provide a novel target to prevent latent infection of B cells with oncogenic gammaherpesviruses.

microbiology↗