Citations

Introduction Uveal melanoma has a marked tropism for the liver where immune tolerance facilitates metastatic progression and resistance to immunotherapy. Impaired CD8 + T cell immunosurveillance is a central determinant of disease progression in uveal melanoma, yet the underlying mechanisms driving liver-mediated immune subpression and hepatic metastasis are not fully understood. Here, we investigate molecular pathways that regulated hepatic CD8 + T cell function and evaluate clinical outcomes associated with liver-directed therapy. Methods Expression of autotaxin (ATX), the expression responsible for generating lysophosphatidic acid (LPA), was evaluated in hepatic resident cell populations. ATX transcript levels were assessed in CD8 + T cells isolated from hepatic metastases and compared with matched peripheral blood and healthy donor control. Antigen-specific effector CD8 + T cells were used to characterize downstream signaling responses to LPA stimulation. Functional studies evaluating cytokine production during chronic antigen exposure were performed using CD8 + T cells from LPA receptor 5-deficient ( Lpar5 -/- ) mice. Results We identify the autotaxin-lysophosphatidic acid (ATX-LPA) axis as a prominent immunoregulatory pathway in the metastatic hepatic tumor microenvironment. ATX, the enzyme responsible for LPA production, is constitutively expressed by hepatic resident cells, is detectable on Kupffer cells and is associated with increased immunosubpressive markers. CD8 + T cells isolated from hepatic metastasis in a patient with uveal melanoma display markedly elevated ATX transcripts compared to matched peripheral blood (>5 log 2 fold change) and healthy controls. Using antigen-specific effector CD8 + T cells, we demonstrate LPA signaling induces low level, persistent ERK phosphorylation distinct from canonical antigen-induced TCR signaling. Genetic deletion of the LPA receptor 5 ( Lpar5 -/- ) on CD8 + T cells restores CD8 T cell percentages and cytokine function during persistent antigen stimulation in the presence of LPA. Discussion These findings define an ATX-LPA mechanisms of dysfunctional ERK signaling that may contribute to hepatic immune subpression in uveal melanoma. We highlight this pathway as a rational therapeutic target alongside liver-directed clinical interventions.

ABSTRACT Introduction Hairy cell leukemia (HCL) is an uncommon hematopoietic stem cell disease known to have an underlying somatic BRAF V600E driver mutation. Currently, the most accepted treatment is five or seven consecutive days of outpatient infusion of cladribine (e.g., continuous infusion), despite frequent antibiotic use and hospitalization with this regimen. As a result, a few investigators have adopted weekly infusion of cladribine with similar or improved results and fewer side effects. Methods We conducted a retrospective, single‐institution cohort study of 36 treatment‐naïve patients with HCL treated at the University of Colorado Hospital. Eighteen patients received intermittent weekly cladribine (intermittent cladribine [IC]) for 5–7 doses and 18 received standard daily cladribine (continuous cladribine [CC]) over 5–7 days. We report clinical cohort characteristics, response rates, progression‐free survival, overall survival, toxicity, hospitalization rates, and measurable residual disease monitoring in select patients using peripheral blood quantitative BRAF V600E PCR. Results We report here our experience with 18 patients with newly diagnosed BRAF ‐mutated HCL treated with weekly cladribine at a single institution compared to 18 HCL patients who received continuous infusions. Baseline hematologic parameters and overall survival were comparable between groups ( p = 0.1135), but there was a higher rate of complete remission for patients treated with IC (94.4% vs. 61.1%) with significantly improved progression‐free survival ( p < 0.0001). We observed comparable rates of neutropenia, neutropenic fever, antibiotic use, and G‐CSF administration. There were fewer hospitalizations for patients treated with IC (4/18) compared to patients treated with CC (6/18). Rituximab exposure differed between groups, with 72% of patients in the IC cohort receiving rituximab compared with 27.2% in the CC cohort (although treatment status was documented for only 66.7% of patients in the CC cohort). Peripheral blood BRAF V600E allele burden significantly declined and correlated with clinical remission, but this was only performed in select patients who were treated in recent years. Conclusions In this retrospective analysis, IC dosed weekly was associated with improved progression‐free survival and higher rates of complete remission compared to CC with comparable toxicity and fewer hospitalizations. These findings suggest a potential clinical benefit from this regimen. However, interpretation of these findings is limited by the non‐randomized design and differences in rituximab exposure between groups, which may have contributed to the observed outcomes and limited definitive conclusions. Trial Registration: The authors have confirmed clinical trial registration is not needed for this submission

Abstract Chimeric antigen receptor (CAR) T-cell therapy achieves high response rates in relapsed/refractory aggressive B-cell non-Hodgkin lymphoma, yet only 40% of patients achieve durable remission. Identifying biological programs linked to long-term response remains critical. As metabolic fitness underlies T-cell persistence and effector function, we profiled metabolic pathways associated with clinical outcomes following commercial CD19 CAR T-cell therapy. Single-cell RNA sequencing of peripheral blood mononuclear cells collected at baseline (BL), peak CAR-T expansion (PK), and one-month post-infusion (M1) was analyzed using CellRanger v7.0.1, immunopipe. Seurat v4.3.0 was applied for unsupervised clustering to delineate cell subsets based on top differentially expressed genes. Patients were categorized as durable complete remission (CR ≥6 months; n = 16), primary refractory (PD1; n = 4), or relapse after initial response (PD2; n = 12). Gene-set enrichment analysis defined metabolic pathway activity across T-cell, monocyte, dendritic cell (DC), and natural killer (NK) subsets. Oxidative phosphorylation (OXPHOS) emerged as the dominant metabolic program distinguishing clinical outcomes. At BL and PK, OXPHOS was consistently enriched in PD1 relative to CR across T-cell, monocyte, and DC subsets, suggesting early oxidative activation in non-responders. By M1, this pattern inverted, with higher OXPHOS activity in CR. PD2 largely paralleled PD1, but several subsets including CD8 T central memory, classical monocytes TGFβ, intermediate monocytes CD38, monocytic myeloid-derived suppressor cell (mMDSC) HIF1A, and mMDSC SIRPA, showed OXPHOS enrichment in CR at BL and/or PK that persisted through M1. In contrast, DC and NK subsets exhibited the opposite pattern: OXPHOS was enriched in CR at PK (conventional DC 2 (cDC2), plasmacytoid DC (pDC), NK, proliferating NK), but shifted toward enrichment in PD2 at M1 (cDC2, NK, NK CD56bright). Glycolysis (GLY) followed a similar trajectory in monocytes and NK cells, with enrichment in PD1/PD2 at BL and PK, followed by enrichment in CR at M1. In T-cells, PD1 maintained GLY enrichment from BL through PK, with no significant differences observed at M1. The inositol-phosphate metabolism pathway showed a more static pattern and was consistently enriched in multiple PD2 effector and memory T-cell subsets without reversal at M1. Taken together, these data reveal distinct, lineage-specific metabolic signatures that differentiate durable remission from early and late progression. Dynamic OXPHOS and GLY programming, characterized by lower activity early and enhanced activity at M1 in CR, may reflect adaptive metabolic programming that supports sustained antitumor immunity. Immune-metabolic profiling may therefore serve as a biomarker of response and highlight actionable metabolic pathways to enhance CAR T-cell durability. Citation Format: Melinda S.Y. Tan, Panwen Wang, Patrizia Mondello, Jacqueline Turner, Andre de Menezes Silva Corraes, Chen Wu, Zuoyi Shao, Kevin Regan, Ma Audrey, Arushi Khurana, Nora N. Benanni, Yucai Wang, Paul Hampel, Jonas Paludo, Saad J. Kenderian, Urshila Durani, Patrick B. Johnston, Jose Caetano Villasboas, Stephen M. Ansell, Ying Li, Haidong Dong, Hu Zeng, Yi Lin. Metabolic pathway signatures defining response to CD19 CAR T-Cell therapy in aggressive B-cell non-hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3278.

Abstract Introduction: Therapeutic outcomes in B-cell non-Hodgkin’s lymphoma (B-NHL) have substantially improved with the introduction of chimeric antigen receptor T-cell (CAR-T) therapy. Yet, persistent immune dysfunction and underlying metabolic dysregulation remain major obstacles to achieving durable clinical responses in B-NHL. We hypothesize that mitochondrial pathways play a critical role in mediating anti-tumor immunity during CAR-T therapy. Methods: Peripheral blood mononuclear cells were collected from healthy controls (CNTRL, n=5) and patients with advanced stage lymphoma (LYM) who received FDA approved CAR-T (n=32). Samples were collected before lymphodepletion (BL), at peak CAR-T expansion (PK) and one-month post-infusion (M1). Single-cell RNA sequencing was used to interrogate differential mitochondrial gene expression. Samples were obtained from LYM patients with complete remission for &gt;6 months (CR), primary refractory (PD1), or relapsed (PD2) disease. Results: Compared to CNTRLs, LYM patients exhibited reduced expression of cytochrome c oxidase. At BL, CR patients demonstrated broader mitochondrial differential gene expression across all T-cell subsets relative to PD1 and PD2 and showed higher expression of MT-ATP-6, MT-ATP-8, and MT-CO3. Following CAR-T infusion, global mitochondrial gene expression increased across patients corresponding with expansion of CD8 effector T cells (Tem tumor circulating) and activated peripheral memory T cells (Tpm). Certain mitochondrial genes, such as MT-CO1, were durably expressed across LYM patients at BL, PK, and M1 in peripheral and central memory T cells (Tpm, Tcm). At PK, cytochrome c oxidase and ATP synthase genes were upregulated in CR Tpm and Tcm. At M1, MT-ATP-6, MT-ATP-8, MT-CO3 were significantly upregulated in CD8 memory precursor effector T-cells (MPECs) of CR patients compared to both PD1 and PD2. While CR and PD2 patients appeared clinically responsive at M1, CR samples maintained significantly higher MT-ATP-8 expression across T cell subsets (CD8 MPECS, Tpm, Tcm) compared to PD2 (p values 0.008; &lt;0.0001; &lt;0.0001). Conclusions: We report a temporal shift in immunometabolism with CAR-T therapy in LYM. Clinical responders exhibited sustained mitochondrial activity characterized by persistent expression of ATP synthase in CD8 MPECs and cytochrome c oxidase in Tpm and Tcm. In sum, mitochondrial programming may distinguish effective versus dysfunctional T cells and identify targets for therapeutic development. Citation Format: Jacqueline Turner, Panwen Wang, Patrizia Mondello, Melinda Tan, Christoph Schaefers, Chen Wu, Andre de Menezes Silva Corraes, Kevin Regan, Zuoyi Shao, Ma Audrey, Arushi Khurana, Nora N. Benanni, Yucai Wang, Paul Hampel, Saad J. Kenderian, Jonas Paludo, Urshila Durani, Patrick B. Johnston, Jose Caetano Villasboas, Stephen M. Ansell, Haidong Dong, Ying Li, Zeng Hu, Yi Lin. Mitochondrial transcriptome profiles associated with clinical responses to CAR-T therapy in aggressive lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1213.

Abstract Increased absolute lymphocyte count (ALC) may predict risk of treatment-related mortality and atypical neurologic events among patients receiving chimeric antigen receptor (CAR) T-cell therapies for relapsed/refractory multiple myeloma. In this study, we analyzed the clinical outcomes of patients receiving ciltacabtagene autoleucel (cilta-cel) at the Colorado Blood Cancer Institute, from September 2023 to January 2025. Baseline data were collected pre/post-CAR T-cell therapy. Patients were stratified into preintervention (treated September 2023 to July 2024) and intervention (treated August 2024 to January 2025; those with ALC &gt;5 × 103/μL received 3 days of dexamethasone prophylaxis on first identification of elevated ALC). In the preintervention group, 9 of 30 patients had peak ALC &gt;5 × 103/μL; 5 of 9 (55.6%) experienced atypical neurologic events and all 5 died (cilta-cel therapy-related complications). In the intervention group, 7 of 23 patients had peak ALC &gt;5 × 103/μL and received dexamethasone; 1 of 7 had an atypical neurologic event; and 1 death occured (infectious complication, 9 months posttreatment). Dexamethasone prophylaxis in patients with ALC of &gt;5 × 103/μL resulted in rapid ALC reduction. Overall survival (OS) was significantly lower in preintervention patients with ALC of &gt;5 × 103/μL vs intervention patients with ALC of &gt;5 × 103/μL and patients with ALC of ≤5 × 103/μL (P = .0013). ALC &gt;5 × 103/μL (vs ALC ≤ 5 × 103/μL) was significantly associated with atypical neurologic events (odds ratio, 6.8; P = .0157) and lower OS (hazard ratio, 6.2; P = .0106). In conclusion, ALC &gt;5 × 103/μL after cilta-cel treatment predicted severe early neurologic events and high mortality risk. Dexamethasone prophylaxis demonstrated promise for risk mitigation.

Abstract Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable response rates in relapsed/refractory aggressive B-cell non-Hodgkin's lymphoma. However, durable remissions occur in only 40% of treated patients, highlighting mechanisms of therapeutic resistance and immune escape. While cytotoxicity is principally executed by CAR T-cells, monocytes shape CAR-T cell efficacy and orchestrate cytokine-driven immunity. In this study, we defined distinct metabolic profiles of T-cell and monocyte subsets based on clinical response. Single-cell RNA sequencing (scRNA-seq) and T-cell receptor sequencing (scTCR-seq) were performed on peripheral blood mononuclear cells at three timepoints: before lymphodepletion (BL), at peak CAR-T expansion (PK), and one-month post-infusion (M1). Data were processed with CellRanger v7.0.1 and analyzed with the immunopipe pipeline (Wang et al., 2025). Seurat v4.3.0 was utilized for unsupervised hierarchical clustering analysis; subsets identified included classical monocytes (CM), intermediate monocytes (IM), non-classical monocytes (NM), and monocytic myeloid derived suppressor cells (mMDSC). Metabolic profiles were compared across patients achieving ≥6-month complete remission (CR, n=16), primary refractory disease (PD1, n=4), or relapsed after a prior response (PD2, n=12). A total of 107035 T cells and 129981 monocytes from 77 serial samples in 32 patients were analyzed. CR patients had a higher T-cell:monocyte ratio than PD1/PD2 at all time points, especially at BL and PK (BL 1.2 vs 0.4, p=0.011; PK 1.3 vs 0.3, p=0.027). Clinical responses were compared with metabolic pathway analysis and identified oxidative phosphorylation (OXPHOS) as the dominant pathway. OXPHOS was consistently enriched in PD cohorts across T-cell subsets, particularly in PD1 vs CR at BL (mucosal-associated invariant T-cell (MAIT) and T peripheral memory (Tpm)), and at PK (CD4/CD8 memory precursor effector cells (MPEC) and activated MPECs (MPECa), CD4 T-helper 1 proliferating (Th1p), CD4 Th2 activated (Th2a), regulatory T-cell (Treg), CD8 naive senescent (CD8NS), CD8 T effector memory (Tem) tumor recirculating (TR), CD8 Tpm, CD8 T resident memory (Trm) TR and MAIT Tpm). By M1, OXPHOS enrichment in CD4/CD8MPECa, CD4Th2a, CD8NS, CD8TemTR reversed, now predominant in CR although CD8Tpm and CD8TrmTR remained enriched in PD1. In the monocyte subsets at BL, PD2 showed OXPHOS enrichment in CD163+ monocytes (mono), IM IL1, mMDSC SIRPA, mMDSC TGFβ, and CM IL6 subsets compared to PD1 and CR, while PD1 showed OXPHOS enrichment in IM CD38, CM TGFβ, and mMDSC HIF1A vs CR and PD2. This persisted at PK. By M1, the pattern reversed and mirrored that of T-cells, with CR patients showing OXPHOS enrichment in CD163+mono, CM IL6, CM TGFβ, IM CD38, mMDSC HIF1A, mMDSC SIRPA and mMDSC TGFβ, and additionally in CM EMT, CM p53, NM, NM CD36 vs PD1/PD2. Glycolysis/gluconeogenesis (GLY) was also enriched across subsets. In T-cells, PD1 vs CR showed GLY enrichment in CD4Th2a, CD8Tpm, CD8Tpm proliferating and MAIT Tpm at PK. In monocytes, GLY was enriched in PD1/2 vs CR at BL (CD163+mono, CM IL6) and PK (CD163+mono, IM CD18, mMDSC SIRPA). By M1, GLY metabolism was enriched in CR vs PD1/PD2 in CD163+mono, CM IL6, mMDSC SIRPA. Additional GLY enriched subsets at CR vs PD1 included CM, CM p53, EMT, IM CD38, IM IL1, mMDSC HIF1A, NM, NM CD36. Inositol phosphate metabolism (IP) was predominantly enriched in PD2 vs CR at BL (CD8NS, CD8Tcm, CD8Tpm activated), PK (CD4Th2, CD8NS, CD8Tem TR, CD8Tpm proliferating, γδT Tem) and M1 (CD4/CD8 MPECa, CD4 Th1/Th2, CD8NS, CD8Tpm, CD8 Tpm proliferating, Treg and MAIT Tpm). Notably, Treg was the only T-cell subset enriched for IP at PK in CR vs PD1/PD2. Additionally, the TCA cycle was enriched in PD1 Tregs and Th2 cells at BL and PK vs CR/PD2. A higher T-cell:monocyte ratio in CR vs PD1/PD2 supports prior evidence that elevated pre-lymphodepletion lymphocyte:monocyte ratios predict better outcomes. We identified distinct metabolic signatures in T-cells and monocytes between CR and PD1/PD2. OXPHOS enrichment in T-cells and monocytes at M1 in CR patients may reflect oxidative programming as a hallmark of sustained anti-tumor immunity. Conversely, GLY enrichment in PD1 T-cells at PK may reflect short-lived effector states lacking persistence. Overall, these findings highlight metabolic profiling as a promising predictive biomarker and therapeutic programming target to enhance CAR T-cell outcomes.