Abstract Antigen density critically influences CAR T-cell efficacy. We show that ibrutinib induces CD19 upregulation in B-cell lymphoma models, enhancing CAR T-cell cytotoxicity. Pharmacologic modulation of target antigen expression represents a promising strategy to overcome resistance.

ABSTRACT:Resistance to Bruton tyrosine kinase (BTK) inhibitors remains a major clinical challenge in B-cell lymphomas and often occurs in the absence of BTK or PLCG2 mutations. Here, we investigated nongenetic mechanisms of ibrutinib resistance in marginal zone lymphoma (MZL) and their broader therapeutic implications. Continuous ibrutinib exposure generated a resistant MZL model that showed cross-resistance to BTK inhibitors and degraders, without evidence of multidrug resistance or genetic alterations. Integrated transcriptomic, epigenetic, and proteomic analyses revealed extensive reprogramming, including the activation of phosphatidylinositol 3-kinase (PI3K)/AKT, MAPK, and MYC pathways, repression of apoptosis and oxidative phosphorylation, and a prominent cytokine-secretory phenotype. Interleukin-16 (IL-16) emerged as a central mediator of resistance. IL-16 was transcriptionally upregulated, actively secreted, and sufficient to induce ibrutinib resistance across multiple CD9+ models of MZL, mantle cell lymphoma, chronic lymphocytic leukemia (CLL), and activated B-cell-like diffuse large B-cell lymphoma. Serum IL-16 levels were elevated in patients with ibrutinib-refractory CLL without BTK/PLCG2 mutations. Mechanistically, IL-16 engaged CD9-enriched membrane microdomains to activate PI3Kδ, thereby sustaining AKT and extracellular signal-regulated kinase signaling, stabilizing MYC, inducing NF-κB-dependent programs, and upregulating antiapoptotic effectors, including BFL1 (BCL2A1). Pharmacological or genetic disruption of the IL-16/CD9/PI3K axis restored sensitivity to BTK inhibitors and R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine [Oncovin], and prednisone) and abrogated IL-16-induced signaling in primary CLL samples. In conclusion, an IL-16/CD9-driven, epigenetically regulated survival pathway represents one possible mechanism of resistance to BTK inhibitors and chemoimmunotherapy, supporting therapeutic targeting of this axis in refractory B-cell lymphomas.

After the introduction of tyrosine kinase inhibitors (TKIs), the number of patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) for chronic phase chronic myeloid leukemia (CP-CML) has dramatically decreased. Imatinib was the first TKI introduced into the clinical arena, predominantly used in the first-line setting. In cases of insufficient response, resistance, or intolerance, patients with CML can subsequently be treated with a second-, third-, or fourth-generation TKI. However, despite the approval of first-, second-, third-, and fourthgeneration TKIs, allo-HSCT still remains indicated for a minority of patients with CML. Here, we discuss the indications in the era of TKIs through different cases representing the clinical situations for which allo-HSCT remains the best option. We also propose our transplant strategy to decrease transplant-related morbidity, particularly graftversus-host disease, and mortality in the particular context of CML, a disease that is one of the most sensitive to immune cellular therapy, allowing the use of a combination of donor lymphocyte infusion and TKIs for posttransplant molecular progression.

Background:The impact of pretransplant cytomegalovirus (CMV) serology and DNAemia on posttransplant clinically significant CMV infections (csCMVi) in allogeneic hematopoietic cell transplant recipients (allo-HCTR) is poorly described. Methods:We performed a single-center cohort study of adult allo-HCTR (16 November 2015-31 December 2023). Letermovir prophylaxis was administered after 01 May 2019 during 100 days (d) posttransplant in CMV-seropositive patients (R+). We investigated associations of pretransplant CMV-IgG-titers and CMV-DNAemia with posttransplant csCMVi and CMV-DNAemia during 6 months posttransplant. In CMVR+, CMV-IgG-titers were classified as "low" (<40 IU/mL) or "high" (≥40 IU/mL). Quantitative CMV-PCR was performed pre-HCT, weekly for 3 months and as clinically indicated. Results:Among the 485 patients included, 209 and 276 underwent a first allo-HCT in the pre- and postletermovir periods, respectively; 314/485 (64.7%) patients were CMVR + . Patients with high CMV-IgG-titers had a higher incidence of csCMVi by d180 posttransplant (47.6%, 95% CI: 41.4-53.6) than those with low CMV-IgG-titers (22.5%, 95% CI: 11.5-35.7) and CMVR- (3.6%, 95% CI: 1.5-7.2, P < .001), and higher incidence of any CMV-DNAemia (P < .001). Overall, 61/485 (12.6%) patients had detectable/quantifiable pretransplant CMV-DNAemia, with higher posttransplant incidence of csCMVi by d180 (61.3%, 95% CI: 47.6-72.4) compared to those with undetectable pretransplant CMV-DNAemia (26.9%, 95% CI: 22.3-31.6, P < .001) and higher incidence of any CMV-DNAemia (P < .001). Pretransplant high CMV-IgG-titers (aHR: 14.18, P < .001) and CMV-DNAemia (aHR: 2.43, P < .001) were strong predictors of posttransplant csCMVi. Conclusions:Pretransplant high CMV-IgG-titers and detectable/quantifiable DNAemia were associated with higher posttransplant csCMVi/CMV-DNAemia incidence, including in the letermovir era. Additional studies on the clinical utility of baseline pretransplant CMV burden in CMV prophylaxis stratification algorithms are needed.

Acute myeloid leukemia (AML) is a highly aggressive hematologic malignancy with a five-year overall survival rate of approximately 30%. AML disrupts normal hematopoiesis and impairs immune response, resulting in a profoundly immunosuppressive tumor microenvironment (TME) that contributes to rapid disease progression and high relapse rates. While chimeric antigen receptor (CAR) T cell therapy has shown promise in B cell malignancies, its efficacy in AML has been limited due to antigen heterogeneity, immune evasion, and the risks of rejection and graft-versus-host disease (GVHD) associated with allogeneic cells. Invariant natural killer T (iNKT) cells are a rare population of innate-like T cells that recognize glycolipids presented by CD1d, which is frequently expressed on AML cells and tumor-associated macrophages. iNKT cells mediate antitumor activity both directly, via CD1d recognition, and indirectly, through immune modulation, such as cross-priming CD8⁺ dendritic cells. Importantly, iNKT cells do not induce GVHD and have been shown to protect against it in preclinical models from studies in our group and others. Moreover, CAR-engineered iNKT cells have demonstrated potent antitumor effects in xenograft models. These unique properties highlight the potential of iNKT cells for allogeneic cell therapy. In this study, we developed allogeneic CD117-targeted CAR-iNKT cells as a potential off-the-shelf immunotherapy for AML. Using immunocompetent C57BL/6 mouse models of spontaneous MLL-AF9 and MLL-ENL AML, we evaluated both the cytotoxic and immunomodulatory functions of iNKT cells in a clinically relevant setting. CD117 CAR-T and CAR-iNKT cells derived from FVB mice exhibited significantly enhanced cytotoxicity in vitro compared to untransduced controls. Notably, our findings demonstrated that CAR-iNKT cells specifically targeted and eliminated CD117high AML cells. In vivo, adoptive transfer of CD117 CAR-T and CAR-iNKT cells modestly extended survival in aggressive AML models (AF9 alone, median 23 days; allogenic T, median 26 days; allogenic CART, median 32.5 days; allogenic iNKT, median 27 days; allogenic CAR iNKT, median 30.5 days; allogenic CART vs T, p = 0.001; allogenic CAR-iNKT vs iNKT, p = 0.0145). We observed functional exhaustion in CAR-iNKT cells following extensive in vitro engineering. Incorporation of murine IL-15 into the CAR construct significantly mitigated exhaustion and improved both persistence and antitumor efficacy in vivo (AF9 alone, median 31.5 days; allogenic CAR iNKT, median 44 days; allogenic CAR iNKT-IL15, median 85 days; allogenic CAR-iNKT vs CAR-iNKT, p = 0.0029). Furthermore, CAR-iNKT cells were capable of eliminating CD1d⁺ tumor-associated macrophages in the spleen and bone marrow, thereby contributing to the reversal of TME-mediated immunosuppression. Together, these findings support CD117 CAR-iNKT cells as a promising and safe allogeneic immunotherapy for AML.

Table S15: Relative quantification by lipid class

Adoptive cell therapies (ACTs), such as chimeric antigen receptor (CAR)-T cell therapy, have revolutionized cancer treatment, especially for hematological cancers. However, patient responses vary considerably. Emerging research reveals a striking influence of time of day (ToD) on ACT efficacy. Administering ACT during the early behavioral active phase enhances tumor control and reduces toxicity in preclinical models, an effect linked to the circadian clock. Latest clinical data also point to ToD effects in the cancer setting. In this opinion article we explore current insights and discuss the emerging underlying mechanisms. We propose that integrating ToD into clinical practice could represent a powerful yet easily implementable therapeutic regimen to improve efficacy and safety of ACT.

Unmanipulated donor lymphocyte infusions (DLI) are crucial for enhancing the graft versus tumor (GVT) effect in post-transplant settings. Practices regarding DLI use vary widely among centers, encompassing differences in indications, prerequisites, and application methods. To explore current DLI policies, we developed a comprehensive survey that garnered responses from 165 EBMT centers across 43 countries. Notably, 97% of respondents reported using DLI in their practices. Indications for DLI included preemptive use for minimal residual disease (MRD) positivity in 86.9% of centers and mixed chimerism in 73.1%; therapeutic use for hematological relapse in 73.1%; and prophylactic use for high-risk disease in 43.8%. Active graft-versus-host disease (GVHD) and active infections were deemed absolute contraindications by 85.6% and 57.5% of centers, respectively. 35% of centers did not consider a prior history of acute (a)GVHD as an exclusion criterion. The majority (71.9%) requested immunosuppression withdrawal before DLI. Most centers (71.3%) collected DLI post-transplant, with 78.1% utilizing unstimulated apheresis. The cell doses applied at the first DLI varied significantly, depending on indication, timing, and donor type. This survey provides the largest overview of current DLI practices, highlighting the need for high-quality data to assess the risks and benefits of different approaches.

Table S12: gene signatures used to analyze RNA-seq data from patients with AML before/after GILT

Table S2: EnrichR analysis of FLT3 gene signatures

Supplementary materials and methods

Table S4: GSEA results in FLT3-ITD PDX AML cells ex vivo-treated with QUIZ or Veh.

Table S8: Multimodal analysis of shCEBPA versus shCTL MOLM14 cell line

B cell receptor (BCR) signaling plays a central role in the pathogenesis of B cell lymphomas, making it a crucial therapeutic target. The advent of BCR-targeted inhibitors, particularly those directed at PI3K and BTK, has revolutionized treatment for B cell non-Hodgkin lymphoma (B-NHL). However, therapeutic resistance remains a significant clinical challenge. Increasing evidence suggests that the tumor microenvironment (TME), particularly the bone marrow (BM) microenvironment, is crucial in driving cancer progression and therapeutic resistance. The BM microenvironment provides a specialized niche where lymphoma cells can evade therapy through interactions with stromal cells and extracellular matrix (ECM) components. Bone marrow stromal cells (BMSCs) contribute significantly to this resistance. In this study, we developed an in vitro 3D model to better understand B cell lymphoma biology and drug resistance mechanisms. We co-cultured lymphoma cell lines with primary BMSCs in a 3D fibrin gel matrix using a high-throughput and automated system. Our results revealed that BMSCs modulate lymphoma cell growth and reduce their sensitivity to the PI3K inhibitor copanlisib and the BTK inhibitor ibrutinib. Furthermore, this model allowed us to identify IGFBP-3, Serpin E1, and PTX-3 as potential mediators of therapeutic resistance. These findings underscore the value of using 3D co-culture models in preclinical settings to more accurately study drug resistance, as they more closely simulate the BM microenvironment's complexity than traditional 2D models, thus improving the predictive value of drug testing in B cell lymphomas. ### Competing Interest Statement L.C. institutional research funds from Orion; travel grant from HTG; A.A. travel grant from Astra Zeneca, consultant for PentixaPharm; F.B. institutional research funds from ADC Therapeutics, Bayer AG, BeiGene, Floratek Pharma, Helsinn, HTG Molecular Diagnostics, Ideogen AG, Idorsia Pharmaceuticals Ltd., Immagene, ImmunoGen, Menarini Recherche, Nordic Nanovector ASA, Oncternal Therapeutics, Spexis AG; consultancy fee from BIMINI Biotech, Helsinn, Menarini; advisory board fees to institution from Novartis; expert statements provided to HTG Molecular Diagnostics; travel grants from Amgen, Astra Zeneca, Beigene, InnoCare, iOnctura. The other Authors have nothing to disclose.

The variable response to cancer immunotherapies highlights a critical gap in our ability to predict and monitor treatment efficacy. To address this, there is an urgent clinical need for advanced molecular imaging technologies that can noninvasively and precisely assess whole-body immune responses. The OX40 receptor (CD134), a potent costimulatory molecule on T cells, serves as a highly specific marker of T-cell activation, an early and crucial event in immunotherapy efficacy. In this study, we report the development of a human OX40-specific radiotracer based on a clinically evaluated therapeutic-ivuxolimab-and assess its utility for PET imaging of activated T cells in vivo. Methods: Deferoxamine conjugation and 89Zr radiolabeling were optimized for ivuxolimab. In vitro specificity of the resultant tracer, 89Zr-ivuxolimab, was then assessed using primary human T cells and stably transfected human OX40+ (huOX40+) human embryonic kidney 293 (HEK293) cells. In vivo specificity and biodistribution of 89Zr-ivuxolimab were confirmed in subcutaneously implanted huOX40+ HEK293 or parental HEK293 tumor-bearing mice. To evaluate 89Zr-ivuxolimab's utility for detecting T-cell activation in vivo, we used a transgenic human OX40 murine model of acute graft-versus-host disease. Ex vivo gamma counting, autoradiography, and immunohistochemistry were performed to verify tracer-binding specificity. Results: 89Zr-ivuxolimab was reproducibly synthesized and showed significantly increased in vitro binding to activated human T cells versus resting cells (P < 0.0001) and increased binding to huOX40+ HEK293 cells versus HEK293 cells (P < 0.0001). Longitudinal PET/CT imaging of tumor-bearing mice over 5 d revealed markedly higher tracer accumulation in huOX40+ HEK293 tumors compared with HEK293 tumors (P < 0.0001). 89Zr-ivuxolimab successfully detected T-cell activation in the spleen, mesenteric lymph node, and gastrointestinal tract of mice with graft-versus-host disease induced by transgenic murine T cells expressing human OX40, compared with control groups (total body irradiation, P < 0.0001; bone marrow, P < 0.001). Ex vivo gamma counting of tissues, autoradiography, and immunohistochemistry corroborated PET findings and confirmed tracer specificity for OX40. Conclusion: 89Zr-ivuxolimab is a promising radiotracer for clinical translation as an imaging agent for activated T cells. Further investigation of its ability to monitor and predict response to different cancer immunotherapy modalities is warranted.

The biological clock plays a central role in hematopoiesis and immune regulation, making circadian rhythms an increasingly important factor in immunotherapies and cellular therapies such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous work by Hou et al suggested that afternoon graft infusions were associated with a higher risk of acute graft-versus-host disease and worse survival, highlighting a potential role for infusion timing in shaping transplant outcomes.To further investigate this, we performed a retrospective analysis of 368 patients who underwent allo-HSCT at Geneva University Hospitals between 2015 and 2024. Observing that infusion timing patterns at our center differed from those reported by Hou et al, we applied a data-driven approach using receiver operating characteristic analysis, which identified 11:17 as the optimal cut-off for overall survival.Stratification using this threshold revealed that patients receiving grafts before 11:00 had significantly improved 2-year overall survival and lower non-relapse mortality compared with those infused later in the day, with no differences in relapse or engraftment rates.These findings suggest that infusion timing may be an important, under-recognized factor influencing allo-HSCT outcomes. Prospective clinical trials are needed to confirm these observations and explore their applicability across different clinical contexts.