7035 Background: While chimeric antigen receptor (CAR) T cells have shown efficacy in B cell lymphomas, most patients (pts) still relapse. Preclinical studies have shown that inhibiting histone methyltransferase EZH2 prevents T cell exhaustion, promotes a T-cell memory phenotype, and sensitizes immune-based approaches. Methods: We administered the EZH2 inhibitor tazemetostat in pts receiving standard-of-care CART for diffuse large B cell, follicular, or mantle cell lymphoma (DLBCL, FL, MCL). Pts received tazemetostat for ≥7 days prior to apheresis and continued to lymphodepletion. Following CART, pts resumed tazemetostat at count recovery and continued for up to 12 mo (6 mo in pts with complete remission [CR]). Peripheral blood mononuclear cells were collected Day -24 and Day -5. To evaluate the immunomodulatory effects of priming, high parameter flow cytometry, RNA-seq with cell deconvolution, differential gene expression patterning, and functional gene signature scoring using ssGSEA were compared. Results: 13 pts enrolled (7 DLBCL, 5 FL, 1 MCL). Median prior lines of therapy were 2 (1-4), and 85% were refractory to the last therapy. EZH2 mutations were present in 2 pts. 4 pts had TP53 mutation or deletion, 4/7 DLBCL pts had MYC translocation and 3 had transformed disease. Five pts received axi-cel, 5 liso-cel, 2 tisa-cel, and 1 brexu-cel. All successfully completed CART manufacturing. The ORR was 100%, including 77% in CR (DLBCL 71%, FL 100%). At a median follow up of 18 months, 54% remain progression-free, and 77% remain alive. Ten pts (77%) experienced CRS, with 1 grade 3 CRS. 6 pts had transient grade 1-2 ICANS. Grade 3+ neutropenia was seen in 77%. Three pts experienced grade 3 infections. The most common AEs were gastrointestinal and almost exclusively grade 1-2. Comparing baseline and pre-CART samples, pts experienced a 14% and 20% increase in NK cells and CD16+ monocytes (p < 0.03) and a 14% and 53% decrease in Tregs and CD14+ monocytes (p < 0.02). Increases in the expression of functional gene signatures associated with MHC-I antigen presentation (p = 0.010), antigen-specific T cell activation (p = 0.019), and cellular cytotoxicity (p = 0.043) were also observed. Parallel reductions in signatures associated with tolerogenic dendritic cells (p = 0.011), myeloid suppression (p = 0.043), and coagulation (p = 0.030) were also seen. There was no observed negative impact on CART transduction efficiency, activation, or expansion. Conclusions: Addition of the EZH2 inhibitor tazemetostat is associated with high clinical efficacy and peripheral immune remodeling. Increases in lymphocyte activation and cytotoxicity along with decreases in myeloid tolerogenicity and immunosuppression could potentially augment CART expansion and persistence. Additional immune correlates are ongoing, and randomized studies are planned to validate this approach. Clinical trial information: NCT05934838 .

7063 Background: Genetic subtypes of diffuse large B-cell lymphoma (DLBCL) capture biological differences between tumors that influence the response to immunochemotherapy (Schmitz et al., NEJM 2018). However, nearly 40% of DLBCL cases remain unclassified. The addition of gene expression signatures can accelerate classification and inform therapeutic intervention. Methods: We generated single-cell RNA sequencing of 103 DLBCL patient biopsies from Weill Cornell Medicine, New York Presbyterian Hospital, and the National Institutes of Health. We also utilized bulk genomic data from a discovery cohort (n=311, Ennishi et al., J Clin Oncol 2019) and a validation cohort (n=574, Schmitz et al., NEJM 2018). Results: Single cell sequencing allowed us to isolate the malignant B cells and develop genetic subtype signatures. The MCD signature was associated with poor overall survival (p<10 -6 ), as was the BN2 signature within ABC tumors (p<10 -3 ). When applied to unclassified tumors, the subtype signatures identified characteristic genetic alterations including SLC1A5 mutations in MCD (p<10 -5 ), UBE2A mutations in BN2 (p<10 -13 ), C10orf12 truncations and copy number loss in EZB (p<10 -6 ), and SGK1 mutations in ST2 (p<10 -7 ). Moreover, we discovered that most DLBCL tumors (80%) contained two or more genetic subclones (median 2, range 1-5) based on DNA copy number differences. The genetic subclones had distinct phenotypes based on expression of six recurrent gene expression meta-signatures, herein termed themes. The germinal center (GC) B cell, memory B cell, plasma cell, and pan-B cell themes reflect B cell differentiation whereas two other themes – cell cycle and cell growth – reflect proliferative and metabolic states that are independent of the differentiation states. Surprisingly, 23% of DLBCL (24/103) harbored genetic subclones expressing B cell differentiation themes that distinguished them from other malignant cells in the same tumor. The GC B cell theme was associated with a favorable response to R-CHOP chemotherapy (p<0.02), as expected, while the cell growth theme (but not the cell cycle theme) was associated with adverse survival (p<0.02). Conclusions: Our study revealed that genetic subtypes have distinct gene expression signatures. We further demonstrated a role for tumor subclones in generating intra-tumoral biological diversity. We developed signatures of inter and intra-tumoral heterogeneity that are associated with overall survival.

Abstract Diffuse large B-cell lymphomas (DLBCLs) with a dark zone (DZ)-like transcriptional profile correlate with poor outcomes to rituximab-based chemoimmunotherapy, but the mechanisms underlying this resistance remain unclear. We hypothesize that DZ-like DLBCLs retain immune-evasion properties of the physiological germinal center (GC) DZ, contributing to treatment resistance. We investigated the molecular basis of spatial T-cell exclusion in GCs and its relevance to immune resistance in DZ-like lymphomas. Digital Spatial Profiling (DSP) and spatial transcriptomics were performed on DZ and light zone (LZ) regions of 10 tonsil GCs to define DZ gene signatures. DZ transcriptional programs in normal DZ and malignant B cells were enriched for cell cycle checkpoints, DNA damage response, ATR activation, and chromatin compaction pathways, correlating with reduced T-cell infiltration. Given that activation-induced cytidine deaminase (AID) is a known driver of the DZ program, we evaluated its role in T-cell exclusion. We observed persistent DZ signatures in AID-deficient and WT murine GCs, with no associated T-cell infiltration. While AID-high B cells enriched for DZ features, AID-low cells overlapped with ATR activation within the DZ program, suggesting ATR-dependent T-cell exclusion independent of AID mutagenesis. ATR inhibition in DZ-like DLBCL cell lines reversed the DZ spatial signature and increased T-cell attraction in microfluidic co-culture systems. In vivo, ATR inhibition in immunized mice significantly increased overall GC T-cell infiltration, particularly CD8+ T cells within the DZ. Finally, in PDX models, ATR inhibition significantly improved CAR-19-mediated cytotoxicity, with the most pronounced effect in DZ-like DLBCL clones, which otherwise exhibited resistance to CAR-T cell killing. These findings support ATR inhibitors as potential adjuncts to chemoimmunotherapy, immune checkpoint blockade, or CAR-T cell therapy in lymphomas, particularly in lymphomas characterized by immune exclusion. This abstract is included in the 18-ICML Abstract Book, https://doi.org/10.1002/hon.70094_172 Citation Format: Valeria Cancila, Giorgio Bertolazzi, Allison S. Y. Chan, Giovanni Medico, Giulia Bastianello, Gaia Morello, Daniel Paysan, Clemence Lai, Hong Liang, Girija Shenoy, Patrick W. Jaynes, Giovanna Schiavoni, Fabrizio Mattei, Silvia Piconese, Maria V. Revuelta, Francesco Noto, Luca Businaro, Adele De Ninno, Ilenia Cammarata, Fabio Pagni, Saradha Venkatachalapathy, Sabina Sangaletti, Arianna Di Napoli, Giada Cicio, Davide Vacca, Silvia Lonardi, Luisa Lorenzi, Andrés J. M. Ferreri, Beatrice Belmonte, Min Liu, Manikandan Lakshmanan, Michelle S. N. Ong, Zhang Biyan, Tingyi See, Kong-Peng Lam, Gabriele Varano, Mario P. Colombo, Silvio Bicciato, Giorgio Inghirami, Leandro Cerchietti, Maurilio Ponzoni, Roberta Zappasodi, Evelyn Metzger, Joe Beechem, Fabio Facchetti, Marco Foiani, Stefano Casola, Anand D. Jeyasekharan, Claudio Tripodo. Aggressive B-cell lymphomas retain ATR-dependent determinants of T-cell exclusion from the Germinal Center Dark Zone [abstract]. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86(13_Suppl):Abstract nr LT02.

Abstract Recent single-cell transcriptomic studies of follicular lymphoma (FL) have revealed heterogeneity among malignant B (MB) cells and selected components of the tumor microenvironment (TME), but have been constrained by relatively small sample sizes and a lack of spatial context. We performed comprehensive cellular profiling and spatial dissection of over five million cells by integrating single-nucleus RNA sequencing (snRNA-seq) from 182 samples (FL, n = 167; control, n = 15) with single-cell spatial transcriptomics (ST) from 386 samples (FL, n = 365; control, n = 21). After stringent quality control, 1,289,073 and 3,968,427 cells from snRNA-seq and ST data, respectively, were analyzed. Unsupervised clustering of snRNA-seq data identified 58 TME cell subtypes. To extend this classification to the ST dataset, we integrated snRNA-seq and ST data by co-embedding them into a shared principal component space, enabling robust batch correction and cell-type label transfer across modalities. Using this supervised annotation framework, ST data recapitulated 54 TME cell subtypes. MB, proliferating MB (MBprolif), and healthy B (HB) cells were classified using sample-level unsupervised clustering in ST. Macrophages exhibited previously uncharacterized heterogeneity in transcriptomic profiles and spatial distribution. We identified CXCL13-expressing macrophage subtypes with high intra- and peri-follicular abundance, termed follicular macrophages (FMs). Non-negative matrix factorization of intrafollicular cell fractions identified five TME archetypes: FM, follicular stroma, two follicular T, and HB archetypes. The FM archetype was enriched for intrafollicular macrophages, including FMs, and showed a significantly higher proportion of grade 3A/3B cases (53.4%) compared to other archetypes (≤16.5%). It was also associated with a higher frequency of high tumor burden requiring treatment initiation rather than a watch-and-wait (WW) approach. Among patients managed by WW, those with the FM archetype showed significantly shorter event-free survival (EFS) compared to those with non-FM archetypes, whereas this association was not observed among treated patients. The percentage of intrafollicular macrophages (IFMP) showed excellent predictive performance for the FM archetype (area under the curve: 0.90). IFMP calculated in a published multiplex spatial protein imaging dataset from an independent cohort of 242 FL patients validated the associations between high IFMP and both pathology grade 3A/3B and shorter EFS in WW patients. Consistently, spatial neighborhood and cell–cell communication analyses revealed co-localization of FMs and MBprolif cells, forming a distinct niche with activated signaling mediated by FM-derived CXCL13, APRIL, and BAFF. Our integrative multi-modal analysis defines previously unrecognized cellular heterogeneity and spatial architecture in FL and highlights macrophage-enriched follicular ecosystems associated with aggressive clinicopathological features, providing a framework for improved biological understanding and patient management. Citation Format: Yoshiaki Abe, Bijal Thakkar, Atish Kizhakeyil, Ashley Wilson, Andrew L. Feldman, Jared Henderson, Amy Ayers, Sara Borgschatz, R. Andrew Harkins, Priya Lakra, Daisuke Kaji, Jonathon B. Cohen, David Russler-Germain, Eric Mou, Francisco Vega, Jennifer Chapman, Giorgio Inghirami, Carla Casulo, Izidore S. Lossos, Jean L. Koff, Chijioke Nze, Peter Martin, Sergei Syrbu, Kiran Vij, David L. Jaye, James R. Cerhan, Christopher R. Flowers, Anne J. Novak, Richard Burack, Dai Chihara, Mamiko Sakata-Yanagimoto, Joshua Tobin, Xubin Li, Michael R. Green. Spatially resolved microenvironment profiling reveals distinct follicular lymphoma archetypes [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A049.

Current US Food and Drug Administration-approved chimeric antigen receptor (CAR) T cell therapies for B cell leukemias and lymphomas target CD19, which is widely expressed across the B cell lineage, often leading to on-target, off-tumor B cell depletion, prolonged immune suppression, and antigen-negative escape in a subset of patients. In contrast, B cell receptor (BcR) signaling is essential for the survival of most mature B cell neoplasms, and BcRs carrying the immunoglobulin heavy variable gene IGHV4-34 are highly enriched in B cell malignancies compared with normal B cells. Further, self-reactive IGHV4-34+ serum autoantibodies are enriched in aggressive systemic lupus erythematosus (SLE) and other autoimmune diseases. Here, we developed CAR T cells targeting the BcR carrying IGHV4-34 (CART4-34). We found that CART4-34 showed specific cytotoxicity and cytokine secretion toward IGHV4-34+ malignant B cells. In addition, although CD19 was down-regulated upon relapse after treatment with CART19, IGHV4-34+ BcR levels remained intact upon relapse after treatment with CART4-34, suggesting reduced risk of antigen-negative escape. In IGHV4-34+ HBL1 cell line-derived xenograft mouse models, CART4-34 showed robust expansion and antitumor activity comparable to those of CART19. Optimized CAR:BcR binding using shorter CAR hinge domains improved immune synapse morphology and in vivo activity. In addition, we showed that CART4-34 could target human IGHV4-34+ SLE B cells and deplete IGHV4-34+ autoantibodies ex vivo, without targeting healthy B cells or affecting total IgG titers. In conclusion, we developed a CAR T cell product that specifically targets pathogenic B cells in lymphoid malignancies and SLE, offering potential for precision cell therapy for these indications.

The clinical and molecular heterogeneity of diffuse large B cell lymphoma (DLBCL) is incompletely understood. By integrating proteomic, transcriptomic, and genomic data from 478 DLBCL tumors, we identify seven DLBCL proteogenotypes (PGs) reflecting specific pathophysiological features that span known molecular subtypes. PG4 is associated with poor outcome independent of established risk factors such as cell-of-origin, international prognostic index, or genetic features. PG4 contains activated B cell-like and germinal center B cell-like tumors and genetically unclassified cases. It shares a dark-zone-related B cell phenotype and shows enrichment for BTG1 mutations that can activate MYC. Single-cell sequencing and spatial transcriptomics reveal enhanced MYC and TCF3/4 transcriptional activity irrespective of MYC translocations. The PG4 tumor microenvironment is characterized by exhausted CD8+ T cells. Our study identifies common oncogenic themes underlying high-risk DLBCL tumors and provides a proteogenomic framework for future diagnostic and therapeutic approaches.

Genetic and gene expression subtypes of diffuse large B cell lymphoma (DLBCL) have been defined using bulk tumor analysis. To explore their biology, we derived single-cell RNA and ATAC sequencing data from 103 DLBCL biopsies and identified malignant B cells by their non-diploid DNA copy number profiles. Using malignant B cell gene expression, we developed and validated signatures of each DLBCL genetic subtype, revealing their distinctive characters. Most biopsies had genetic subclones, defined by distinct patterns of aneuploidy, that were distinguished by expression of biological themes reflecting B cell differentiation state, cell proliferation, and cell growth. This analysis revealed REL amplification as a mechanism to block terminal memory B cell differentiation. The genetic subtype signatures and biological themes varied independently, had distinctive transcription factor networks, and were associated with survival following chemotherapy. This single-cell resource illuminates intra- and inter-tumoral biological variation, facilitating studies of DLBCL pathogenesis and therapeutic response.

Myelodysplastic neoplasms (MDS) disrupt bone marrow hematopoiesis, yet clinical assessment relies largely on blast enumeration and qualitative morphology, which incompletely capture marrow architecture and disease state. We applied whole-slide multiplex immunofluorescence imaging with single-cell phenotyping to map bone marrow microarchitecture in MDS. Diagnostic biopsies (n = 36), longitudinal treatment samples (n = 29), precursor states (n = 13), and normal controls (n = 21) were analyzed, comprising >5 million spatially resolved cells. MDS marrow exhibited coordinated, genotype-imprinted architectural remodeling, including altered progenitor composition and spatial patterning, disrupted erythroid island organization, and displacement of hematopoietic stem and progenitor cells from perivascular niches. Interrogation of 82 cellular and spatial features yielded a composite Microarchitectural Perturbation Score (MDS-MAPS), derived from diagnostic samples and fixed prior to longitudinal analyses. In leave-one-patient-out cross-validation, MDS-MAPS discriminated remission from active disease more accurately than blast percentage (AUC 0.883 vs 0.660) and distinguished low-blast MDS from clonal cytopenia of undetermined significance (CCUS) (AUC 0.815). Mixed-effects modeling showed MAPS decreased in remission statistically independent of blast burden, with architectural normalization during remission and re-emergence at relapse. These findings define quantitative bone marrow architecture as a dynamic tissue-state biomarker that complements molecular and blast-based assessment in MDS.

Large B-cell lymphomas (LBCL) are a clinically and molecularly diverse group of malignancies with a rapidly evolving therapeutic landscape that has introduced new areas of clinical need, such as post-CD19 chimeric antigen receptor T (CART19) progression. Patient-derived xenograft (PDX) models are an important tool for mechanistic studies and preclinical evaluation of new therapies and can be generated from a variety of clinical contexts that capture tumor-intrinsic resistance mechanisms. We therefore undertook a comprehensive effort to generate PDX models that encompass the molecular landscape of LBCLs and include important clinical scenarios for new drug development. Here, we describe the first 48 models within this publicly available repository, capturing the transcriptional and genetic subsets of LBCL. These models also include 23 generated from post-CART19 progression patient biopsies, which reproduce patterns of progression driven by CD19 mutation or expression loss, as well as tumor cell-intrinsic CART19 resistance that we validated in vivo. SIGNIFICANCE:Here, we describe X-LYMPH (Xenografts of Lymphoma), a publicly available and molecularly annotated PDX repository that captures the heterogeneity of LBCL. X-LYMPH includes models of CAR T-cell resistance, providing a shared foundation for mechanistic research and therapeutic development for lymphomas. See related commentary by Evgin and Steidl, p. 655.

ABSTRACT:Few prospective benchmark studies exist to characterize the evolving contemporary real-world practice for peripheral T-cell lymphoma (PTCL). We report the patterns of first-line care and outcomes for 720 patients with systemic PTCL enrolled in 2 related prospective cohort studies, Lymphoma Epidemiology of Outcomes (LEO) from 2015 to 2020 and Molecular Epidemiology Resource (MER) from 2002 to 2015, both followed to 2024. The primary end points were event-free survival (EFS) and overall survival (OS) using Kaplan-Meier estimator and Cox regression model. Secondary end points included correlations of clinical and treatment factors with survival. The most common induction regimens were CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) based (70%), given as CHOP (36%), CHOP plus etoposide (23%), or CHOP-like plus novel agents (11.5%, including 5% BV-CHP [brentuximab plus cyclophosphamide, doxorubicin, and prednisone]). Consolidative autologous stem cell transplant was performed in 102 patients (14%). Within nodal PTCL, EFS and OS were adversely associated with International Prognostic Index 2 to 5, prognostic index for T-cell lymphoma score 1 to 4, and non-anaplastic large-cell lymphoma (ALCL) subtypes. Within LEO, which captured increasing first-line etoposide and brentuximab vedotin, adding etoposide to CHOP was associated with better OS in anaplastic lymphoma kinase-negative ALCL. BV-CHP showed a trend toward OS improvement in ALCL. Patients failing EFS6 and EFS24 had 5-year subsequent OS of 12% and 17%, respectively. The inferior outcomes in non-ALCL subtypes and patients failing EFS6 and EFS24 highlight unmet needs with CHOP-based induction, where clinical trials with targeted therapy should be prioritized. This trial was registered at www.clinicaltrials.gov as NCT02736357.

Myeloproliferative neoplasms (MPNs) are sustained by mutated hematopoietic stem cells (HSCs). Existing therapies fail to eliminate this compartment, leaving allogeneic HSC transplantation as the only curative option. Recurrent MPN driver mutations in calreticulin ( CALRmut ) generate a C-terminal neopeptide that requires cell-surface expression for oncogenic signaling, making it an attractive immunologic target. However, it remains unknown if CALRmut is uniformly displayed on all MPN HSCs within hematopoietic microenvironments. We generated huAB2, a high-affinity CALRmut-specific humanized antibody, to use as the targeting domain for chimeric antigen receptor (CAR)-T cells. We show that CALRmut is consistently displayed on functional MPN HSCs and accessible in vivo . huAB2 CAR-T cells eradicate MPN-propagating CALRmut HSCs in patient-derived tumor xenograft models without antigen escape while preserving coexisting normal human and host hematopoiesis. These findings establish CALRmut display as an obligate feature of MPN HSC fitness and support the feasibility of curative, non-transplant immunotherapy for CALRmut MPNs. Significance:Therapies that eradicate cancer stem cells enable cure, but their feasibility is unknown. We establish an approach to potentially cure MPNs by proving mutant calreticulin to be a MPN stem cell marker that can be targeted by CAR-T cells to selectively wipe out disease in preclinical models of human MPNs.

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogenous disease, with variable response to T-cell therapies, highlighting the need for a clinically applicable patient stratifier. To dissect DLBCL complex tumor-host immune interactions, we established fully humanized DLBCL PDX models (HuMice PDX) and performed integrated multi-omics profiling of patient samples. Methods: We analyzed 132 DLBCL (112 R-CHOP, 20 CAR-T patients), 48 matched PDX, 9 HuMice PDX and CAR-19/CAR-BAFFR treated PDX, using bulk RNA-seq, single-cell RNA/TCR-seq, WES/CAPP-seq and CosMx SMI profiling (6000 RNA, 64 proteins). Large DLBCL datasets were used to build a prediction classifier. heterologous/allogeneic CD34+ Human Stem Progenitor Cell (HSPC) were implanted in NSG-SGM3xNBSGW mice, to generate HuMice. Results: We identified EPICORE, a 12-gene epigenetic gene-expression signature, able to predict whether DLBCL displays an immune-depleted (ID) versus an immune-rich (IR) tumor microenvironment. ID/EPICORE-high patients displayed significantly worse outcomes following both R-CHOP-(p<0.0001) and CAR-19 (p=0.002) treatment, compared to IR/EPICORE-low patients. To investigate host-lymphoma interactions, we generated HuMice and implanted them with ID/EPICORE-high and IR/EPICORE-low PDX models. Mimicking patients’ outcomes, only IR models displayed reduced tumor growth in HuMice, while ID-DLBCL showed unimpeded growth. Flow cytometry, mIHC, and RNAseq/scRNAseq analyses revealed that ID-DLBCL-HuPDX were devoid of Tumor-Infiltrating Lymphocytes (TILs), whereas IR-DLBCL-HuPDX displayed abundant (TILs) (p<2e-04) and clonal expansion. We then tested two different CART (CD19, BAFFR) in-vivo in nine ID- and IR-PDX-HuMice models. IR/EPICORE-low models were eradicated, while ID/EPICORE-high were refractory to both CAR. To enhance ID-DLBCL responses, we performed drug screening of clinical phase epigenetic targeting drugs shown to improve immune responses. Valemetostat and Lenalidomide treatments were associated with improved CAR-T cell killing, both in-vitro and in-vivo. Exposure to drug led to a reduction of EPICORE score, and enrichment of inflammatory signaling pathways (NFkb, IFNg/a, IFN-dependent chemokines), thereby converting ID-DLBCL into IR-DLBCL (p<0.05-0.0005) Conclusions: EPICORE, a novel classifier, predicts DLBCL T-cell infiltration and therapy outcomes allowing patients stratification. Humanized PDX accurately reflect DLBCL physiopathology and immune-host interactions, providing a platform for validating next-generation immunotherapies. Epigenetic therapies reprogram EPICORE-high/ID- into EPICORE-low/IR-DLBCL, overcoming immune refractoriness and enhancing T-cell infiltration and tumor immunorecognition. Citation Format: Giovanni Medico, Giorgia Zanetti, Giorgio Bertolazzi, Francesco Vallania, Paul Zumbo, Maider Amiama, Doron Betel, Maria Cacciapuoti, Abigail Taylor, Clarisse Kayembe, Sanjay Patel, Arvin Ruiz, Jennifer Ziello, Jason Weirather, Gulpreet Kaur, Luca Cappelli, Luca Paruzzo, Marco Ruella, Zhenyuan Dong, Larry Kwak, Nicolás Di Siervi, Maria Revuelta, Claudio Tripodo, Leandro Cerchietti, Giorgio Inghirami. Humanized PDX models uncover drug-reversible epigenetic immune exclusion governing T-cell therapy response in DLBCL, captured in patients by EPICORE stratifier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB427.

TET2 mutations are frequent in TFH-derived lymphomas, but how epigenetic disruption initiates malignant T cell transformation is unclear. We generated Cd4cre;Tet2FL/FL mice, which developed aggressive T cell lymphoma (m-TCL) with a TFH cell-like immunophenotype. Genome-wide transcriptomics and epigenetic profiling of Tet2-/- CD4+ T cells prior to lymphoma development showed hyperactive TCR, PI3K signaling, and dysregulated TH differentiation program, with proliferation promoting signal transduction at the lymphoma stage. Tet2 loss promoted hyperplasticity under in vitro conditions but favored conditional TFH differentiation. Reduced 5-hmC levels at regulatory genomic elements, resulted in transcriptional rewiring of TFH-associated genes, promoting ICOS(L)-mediated PI3K signaling. TET2-KO human CD4+ T cells showed conserved epigenetic changes with increased proliferation, decreased exhaustion, increased memory marker expression, and clonal expansion with restricted TCR repertoire under in vitro conditions. scRNA-seq revealed a persistent proliferative cluster characterized by elevated stem-like transcriptional features compared with WT counterparts. Tet2-/- m-TCLs allografted into NSG mice showed a significant response to epigenetic (5-azacytidine) and PI3K inhibitors (duvelisib) alone or in combination.

Anaplastic large cell lymphomas (ALCLs) are CD30+ T-cell lymphomas that share pathologic features but differ in presentation, outcome, and genetics. Current classification incorporates clinical presentation and ALK status, but inadequately addresses molecular heterogeneity and therapeutic vulnerabilities. We studied 689 ALCLs in the Lymphoma/Leukemia Molecular Profiling Project and performed expert consensus review, genetic subtyping (ALK, DUSP22, TP63, and triple-negative), and immunohistochemistry for phospho-STAT3Tyr705. RNAseq with unsupervised gene expression profiling in a sub-cohort (N=393) identified two main molecular types of ALCL that could be predicted with 91% accuracy based on the presence (Type I) or absence (Type II) of phospho-STAT3Y705 expression (P<0.0001). Type I ALCLs included ALK+ ALCL and a subset of triple-negative ALCLs (TN-I); Type II ALCLs included tumors with DUSP22 and/or TP63 rearrangements and the remaining triple-negative ALCLs (TN-II). Type I ALCLs were enriched for JAK-STAT3 (FDR<0.0001), whereas Type II ALCLs were enriched for non-tyrosine kinase pathways, particularly epigenetic regulators such as EZH2 (FDR<0.0001). EZH2 and H3K27me3 were overexpressed by immunohistochemistry (P<0.0001). Prognosis in systemic ALCL was favorable for DUSP22-rearranged ALCL (5-year OS, 95%; N=49) and ALK+ ALCL (88%; N=101), intermediate for triple-negative ALCL (TN-I, 52% and TN-II, 37%; N=92), and poor for TP63-rearranged ALCL (0%; P<0.0001; N=15). We introduce an integrated molecular classification that preserves currently diagnosed ALCL entities but identifies four molecularly distinct ALK− ALCL subtypes (DUSP22-rearranged, TP63-rearranged, TN-I, and TN-II). This classification can be easily implemented on paraffin tissue in routine practice or clinical trials and stratifies ALCL into diagnostically, prognostically, biologically, and potentially therapeutically relevant subtypes.

ABSTRACT:This phase 2 study evaluated the efficacy and safety of combining acalabrutinib and lenalidomide with either rituximab (ALR) or obinutuzumab (ALO), with longitudinal minimal residual disease (MRD) monitoring in frontline MCL treatment. The primary objective was molecular complete response (CR) after 12 cycles of induction, defined by Lugano criteria, and undetectable MRD of <10-6 (uMRD6) by clonoSEQ. Secondary objectives included safety, responses, and survival. Exploratory objectives included tumor mutation profiles and cell-free DNA (cfDNA) by cancer personalized profiling by deep sequencing. Patients in uMRD6 molecular CR were eligible for discontinuation of acalabrutinib plus lenalidomide after 24 cycles; all patients received a minimum of 36 cycles of anti-CD20 antibody treatment. In the ALR cohort, grade 3/4 hematologic toxicities included neutropenia (38%), thrombocytopenia (4%), and anemia (4%). Nonhematologic toxicities included rash (42%), fatigue (4%), nausea (4%), and vomiting (4%). The overall response rate (ORR) was 100%, CR rate was 83%, and molecular CR rate was 67% after 12 cycles of induction, with best molecular CR at 83%. At a median follow-up of 53 months (range, 46-60), the 4-year overall survival (OS) and progression-free survival (PFS) for ALR were 91% and 76%, respectively. TP53 mutations were adversely associated with PFS (P = .026). For ALO, ORR, CR, and molecular CR were 90% after induction, and 2-year OS and PFS were both at 100%. Longitudinal cfDNA analysis in ALR revealed clonal evolution during response and progression. This safe and active regimen is feasible as a time-limited initial therapy for patients with MCL and warrants further evaluation in response-adapted strategy. This trial was registered at www.ClinicalTrials.gov as NCT03863184.

ABSTRACT:Resistance to first-line chemotherapies and crizotinib in anaplastic large cell lymphoma (ALCL) represents a significant challenge, often leading to a dismal outcome. Despite recent advancements, the dissection of the intrinsic and extrinsic molecular alterations underlying crizotinib resistance in ALCL is still poorly understood. Here, we transcriptionally unraveled the bidirectional interplay between anaplastic lymphoma kinase (ALK)-driven ALCL (ALK+ ALCL) and stromal cells in the presence of crizotinib at bulk and single-cell levels and identified that the microenvironment provides prosurvival signals leading to crizotinib persistence in ALK+ ALCL. We detected increased B-cell lymphoma 2 (BCL2) expression and downregulation of pathways related to apoptosis in crizotinib-persister ALK+ ALCL cells. Furthermore, we predicted in silico the ligand-receptor interactions between tumoral and stromal cells, supporting their contribution to ALCL pathogenesis mainly participating in the adhesion/membrane transport, triggering receptors, and promoting activation and microenvironment stimulation in lymphoma cells. Finally, we explored the effect of crizotinib in combination with BH3 mimetics. Pharmacologic and genetic ablation of anti-apoptotic targets displayed a significant synergistic effect with crizotinib, overcoming the stroma-mediated protection of lymphoma cells on drug treatment. Thus, BCL2/B-cell lymphoma-extra large (BCL-XL) targeting is synthetic lethal with crizotinib exposure in ALK+ ALCL and represents an intrinsic- and extrinsic-mediated targetable vulnerability in lymphoma cells challenged with crizotinib. Our data support the evaluation of BCL2 targeting in crizotinib-based regimens in the management of patients with ALK+ ALCL.

Cancer progression involves genetic and epigenetic changes that disrupt chromatin 3D organization, affecting enhancer-promoter interactions and promoting growth. Here, we provide an integrative approach, combining chromatin conformation, accessibility, and transcription analysis, validated by in silico and CRISPR-interference screens, to identify relevant 3D topologies in pediatric T cell leukemia (T-ALL and ETP-ALL). We characterize 3D hubs as regulatory centers for oncogenes and disease markers, linking them to biological processes like cell division, inflammation, and stress response. Single-cell mapping reveals heterogeneous gene activation in discrete epigenetic clones, aiding in patient stratification for relapse risk after chemotherapy. Finally, we identify MYB as a 3D hub regulator in leukemia cells and show that the targeting of key regulators leads to hub dissolution, thereby providing a novel and effective anti-leukemic strategy. Overall, our work demonstrates the relevance of studying oncogenic 3D hubs to better understand cancer biology and tumor heterogeneity and to propose novel therapeutic strategies.

The germinal center (GC) dark zone (DZ) and light zone represent distinct anatomical regions in lymphoid tissue where B cell proliferation, immunoglobulin diversification, and selection are coordinated. Diffuse large B cell lymphomas (DLBCLs) with DZ-like gene expression profiles exhibit poor outcomes, though the reasons are unclear and are not directly related to proliferation. Physiological DZs exhibit an exclusion of T cells, prompting exploration of whether T cell paucity contributes to DZ-like DLBCL. We used spatial transcriptomic approaches to achieve higher resolution of T cell spatial heterogeneity in the GC and to derive potential pathways that underlie T cell exclusion. We showed that T cell exclusion from the DZ was linked to DNA damage response (DDR) and chromatin compaction molecular features characterizing the spatial DZ signature, and that these programs were independent of activation-induced cytidine deaminase (AID) activity. As ATR is a key regulator of DDR, we tested its role in the T cell inhibitory DZ transcriptional imprint. ATR inhibition reversed not only the DZ transcriptional signature, but also DZ T cell exclusion in DZ-like DLBCL in vitro microfluidic models and in in vivo samples of murine lymphoid tissue. These findings highlight that ATR activity underpins a physiological scenario of immune silencing. ATR inhibition may reverse the immune-silent state and enhance T cell-based immunotherapy in aggressive lymphomas with GC DZ-like characteristics.