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Kline, J.

Publications and source records attributed to Kline, J..

7 recordsLinked to original sources

Harnessing the PRDM1-PGC1A axis to enhance CAR T cell efficacy

Despite its promise in treating B cell malignancies, chimeric antigen receptor (CAR) T cell therapy remains limited by resistance mechanisms that impair CAR T cell stemness, persistence, and effector function. Here, we identified PRDM1, which encodes the transcriptional repressor, BLIMP-1, as a crucial regulator of CAR T cell terminal differentiation and exhaustion. PRDM1 was highly enriched in exhausted-like tumor-infiltrating CAR T cells from lymphoma patients and in pre-infusion products from clinical non-responders. These findings link increased PRDM1 expression to CAR T cell dysfunction and poor clinical outcomes. Genetic deletion of PRDM1 significantly promoted CAR T cell proliferative capacity, memory differentiation and antitumor efficacy in both hematologic and solid tumor models. Mechanistically, PRDM1 deletion increased the activity of PGC1a;, a transcriptional coactivator central to mitochondrial biogenesis and antioxidant capacity, thereby improving mitochondrial function, oxidative stress resistance, and metabolic reserve. Depletion of PGC1a; attenuated the improved efficacy of PRDM1-deficient CAR T cells, indicating an essential role for this pathway downstream of PRDM1 loss. Pharmacologic activation of the PPAR-PGC1a; program with a clinically approved agonist, bezafibrate, enhanced CAR T cell antitumor activity without direct tumoricidal effects. Notably, transient exposure to bezafibrate during CAR T cell manufacturing was sufficient to confer durable treatment benefits. Together, our study defines a PRDM1-PGC1a; axis that controls CAR T cell differentiation, mitochondrial fitness and therapeutic response, which provides a mechanistic and translational framework for advancing CAR T cell therapy across distinct tumor contexts.

immunology↗

Potentiating CAR-T bystander killing by enhanced Fas/FasL signaling mitigates antigen escape in heterogeneous tumors

Antigen (Ag) escape is a frequent mechanism of relapse after CAR-T therapy, even though only [~]1% of leukemic and [~]0.1% of lymphoma cells are Ag- at baseline. In this study, we modeled extreme Ag heterogeneity (>20%) to define how Fas/FasL-dependent bystander killing contributes to tumor clearance. Across patient cohorts, Fas expression predicted survival after CD19 CAR-T therapy, particularly in CD19-low disease. In both murine and human systems, Fas-dependent bystander killing required Ag stimulation and cell contact, operated within a defined therapeutic window, and could eradicate large fractions of Ag- tumors in vivo. Pharmacologic potentiation with inhibitor of apoptosis protein antagonists or genetic stabilization of CAR-T membrane-bound FasL enhanced bystander killing but simultaneously induced CD4 T cell fratricide, which was rescued by CAR-T Fas knockout. Importantly, Fas sensitization also enabled bispecific antibody-redirected T cells to mediate bystander killing in resistant tumors. Finally, targeting tumor-associated macrophages triggered Fas-dependent clearance of neighboring tumor cells. These findings establish Fas-mediated bystander killing as a generalizable and therapeutically actionable axis to prevent Ag escape and broaden the scope of targeted T cell therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/677496v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@11bedorg.highwire.dtl.DTLVardef@d09d07org.highwire.dtl.DTLVardef@1f53c1dorg.highwire.dtl.DTLVardef@13d96f9_HPS_FORMAT_FIGEXP M_FIG C_FIG Fas-mediated bystander killing can be potentiated by IAP inhibition and FasL stabilization, and further improved by decoupling CD4 T cell fratricide via Fas knockout. Schematic representation of CAR-T bystander killing across three mechanistic states. Left (Baseline): Antigen-positive (CD19) tumor cells activate CAR-T cells, leading to FasL-mediated cytotoxicity of neighboring antigen-negative (CD19-) tumor cells via membrane-bound FasL. Limited FasL expression and ADAM10-mediated shedding constrain bystander killing, and CD4 T cells are largely spared. Middle (Potentiated): Enhancing Fas signaling through IAP inhibition (IAPi), increasing FasL expression, and reducing FasL shedding result in improved bystander killing but also increased CD4 T cell fratricide. Right (Potentiated & Decoupled): Combining IAPi with FasL stabilization and CAR-T Fas knockout (FasKO) augments bystander killing while eliminating CD4 T cell fratricide.

immunology↗

Two-Stage CD8+ CAR T-Cell Differentiation in Patients with Large B-Cell Lymphoma

Chimeric antigen receptor (CAR) T-cell therapy has expanded therapeutic options for patients with diffuse large B-cell lymphoma (DLBCL). However, progress in improving clinical outcomes has been limited by an incomplete understanding of CAR T-cell differentiation in patients. To comprehensively investigate CAR T-cell differentiation in vivo, we performed single-cell, multi-modal, and longitudinal analyses of CD28-costimulated CAR T cells from infusion product and peripheral blood (day 8-28) of patients with DLBCL who were successfully treated with axicabtagene ciloleucel. Here, we show that CD8+ CAR T cells undergo two distinct waves of clonal expansion. The first wave is dominated by CAR T cells with an exhausted-like effector memory phenotype during the peak expansion period (day 8-14). The second wave is dominated by CAR T cells with a terminal effector phenotype during the post-peak persistence period (day 21-28). Importantly, the two waves have distinct ontogeny and are biologically uncoupled. Furthermore, lineage tracing analysis via each CAR T cells endogenous TCR clonotype demonstrates that the two waves originate from different effector precursors in the infusion product. Precursors of the first wave exhibit more effector-like signatures, whereas precursors of the second wave exhibit more stem-like signatures. These findings suggest that pre-infusion heterogeneity mediates the two waves of in vivo clonal expansion. Our findings provide evidence against the intuitive idea that the post-peak contraction in CAR abundance is solely apoptosis or extravasation of short-lived CAR T cells from peak expansion. Rather, our findings demonstrate that CAR T-cell expansion and persistence are mediated by clonally, phenotypically, and ontogenically distinct CAR T-cell populations that serve complementary clinical purposes.

immunology↗

Conserved principles of spatial biology define tumor heterogeneity and response to immunotherapy

The complexity of tumor microenvironments (TMEs) poses a substantial challenge to understanding tumor heterogeneity and clinical outcomes. By studying an ensemble of 262 diverse solid tumors, we uncovered a conserved, hierarchical architecture of transcriptionally covarying regions we term Spatial Groups (SGs). SGs corresponded to discrete biological units as benchmarked against multiple spatial technologies, and their nested organization revealed context-dependent constraints within tumors. Using SGs for comparing tumors, we derived a pantumor classification where immune spatial heterogeneity was the dominant axis of variation. This classification stratified response to immune checkpoint blockade in an out-of-sample cohort of non-small cell lung cancer patients. Statistical approximation techniques defined a sparse set of protein markers capturing system-level properties of TME spatial biology, demonstrating a framework for distilling genome-wide information into clinically deployable diagnostics. Our findings position the architecture of SGs as a general model unifying TME structure with biological function and clinical translation.

cancer biology↗

Integrative genomic analysis identifies unique immune environments associated with immunotherapy response in diffuse large B cell lymphoma

Most diffuse large B-cell lymphoma (DLBCL) patients treated with bispecific antibodies (BsAb) or chimeric antigen receptor (CAR) T cells fail to achieve durable treatment responses, underscoring the need for a deeper understanding of mechanisms that regulate the immune environment and response to treatment. Here, an integrative, multi-omic approach was employed to characterize DLBCL immune environments, which effectively segregated DLBCLs into four quadrants - termed DLBCL-immune quadrants (IQ) - defined by cell-of-origin and immune-related gene set expression scores. Recurrent genomic alterations were enriched in each IQ, suggesting that lymphoma cell-intrinsic alterations contribute to orchestrating unique DLBCL immune environments. In relapsed/refractory DLBCL patients, DLBCL-IQ assignment correlated significantly with clinical benefit with the CD20 x CD3 BsAb, mosunetuzumab, but not with CD19-directed CAR T cells. DLBCL-IQ provides a new framework to conceptualize the DLBCL immune landscape and uncovers the differential impact of the endogenous immune environment on outcomes to BsAb and CAR T cell treatment.

cancer biology↗

A novel multifunctional role for Hsp70 in binding post-translational modifications on clients

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and inability of current technologies to distinguish direct vs bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the budding yeast Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multi-point interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors which can be used to probe chaperone function in cells, we have also identified a suite of PTM-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically-important PTMs on client proteins. O_LIIn vivo confirmation of Hsp70 dimerization C_LIO_LIComprehensive direct interactome of Hsp70 C_LIO_LIMulti-domain interactions between Hsp70 and client proteins C_LIO_LIIdentification of novel biologically-important client protein PTMs C_LI

molecular biology↗

Identification and characterization of novel proteins from Arizona bark scorpion venom that inhibit Nav1.8, a voltage-gated sodium channel regulator of pain signaling

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes 8 cysteines and a "CXCE" motif where X = an aromatic residue (tryptophan, tyrosine or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

pharmacology and toxicology↗