Abstract Background. Commanding kinase selectivity remains a major bottleneck in the discovery of targeted anticancer drugs, with homologous ATP-binding pockets leading to off-target inhibition, toxicity, and reduced clinical effectiveness. Addressing these difficulties, we present a streamlined computational workflow allowing rationalizing selective compound activity by capturing subtle, transient structural differences across many homologous kinases. We demonstrate our approach by characterizing the diverging mechanisms of action of nemtabrutinib and ibrutinib in cell lines derived from two specific B-cell lymphoma subtypes (activated B-cell like [ABC] and germinal-center B-cell like [GCB] DLBCL), correlating in vitro anti-tumor activity with in silico selectivity scores computed across 20 tyrosine kinases. Methods. To enable high-throughput, cost-efficient parallel processing of ATP-binding pocket variants, as a preliminary step we compiled a subset of metastable pocket conformations adopted by tyrosine kinases (DFG motif and associated dihedral) with and without generic compound binding (templating with AlphaFold2 and publicly available data). The structural alignment of this conformational dictionary and its processing via structure-based pharmacophore methods (CDP:Kit) produced rich maps of kinase pocket interactomes. Fitting compounds into these maps then enabled scoring matching pharmacophore features and computing a relative selectivity score between kinases for each compound (CDP:Kit and lightweight binding energy estimations) using BTK as baseline for confirmed targeting. Results. We deciphered the synergistic interplay of 10 residue locations within the extended ATP-binding pocket, responsible for the selective binding of nemtabrutinib and ibrutinib across tyrosine kinases. As aligned to human BTK sequence and specifying potential for modified interactions, we highlight reference locations: Q412 (loop-mediated covalent binding, none), V416 (hydrophobic, H-bond), F442 and M449 (hydrophobic, sulfur-aromatic, cation-pi), L460 and I472 (hydrophobic, sulfur-aromatic, pi-stacking), T474 (hydrophobic, halogen-bond, H-bond, sulfur-aromatic, pi-stacking), C481 (covalent binding, H-bond, none), N484 (H-bond, none) and L542 (hydrophobic, sulfur-aromatic, none). Conclusions. We demonstrate a general strategy for rational, structure-guided characterization of selective compound activity across conserved kinase families. Our approach enabled characterizing the diverging binding modes of nemtabrutinib and ibrutinib across 20 kinases, in agreement with anti-tumor activity data and expression levels in cell lines derived from two B-cell lymphoma subtypes. We identified FYN, FRK and MAST1 as likely targets of nemtabrutinib and not of Ibrutinib, responsible for GCB DLBCL non-proliferation. Citation Format: Charly Empereur-mot, Giulio Sartori, Luca Pesce, Filippo Spriano, Chiara Tarantelli, Luciano Cascione, Alberto J. Arribas, Luca Aresu, Davide Rossi, Giovanna L.M. Damia, Massimo Broggini, Daniela Polino, Francesco Bertoni. Pharmacophore profiling highlights diverging binding modes of nemtabrutinib and ibrutinib across tyrosine kinases [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 982.

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.

Supplementary Fig. S1. Structure and characterization of ADCT-602. (A) Full-length amino acid sequence of light and heavy chains of hLL2-C220, with the mutations C219S in the light chain and C225V and C228V in the heavy chain indicated in bold. (B) Structure of ADCT-602 with the PBD dimer payload tesirine (SG3249) conjugated at C219 of hLL2-CC20 (C220 according to the EU numbering system) in the heavy chain. (C) ADCT-602 characterized by size exclusion chromatography and by (D) reduced reversed-phase chromatography. L indicates elution of the light chain, whereas H0 and H1 indicate elution of the unconjugated H-chain or H-chain conjugated to SG3249, respectively. (E) RP-HPLC analysis of the reduced tryptic digest of 2 batches of ADCT-602 (red and black traces). Ala alanine; PABC poly(A)-binding protein C-terminal; PBD pyrrolobenzodiazepine; Val valine.

Abstract Background. Olfactory receptors (ORs) are a large subgroup of chemosensors within the G protein-coupled receptor (GPCR) family and have been found ectopically throughout the human body, where they fulfil a range of functions and are associated with the pathogenesis of numerous diseases. While certain ORs have been linked to cancer development, evidence regarding their role in hematological diseases remains scarce. OR13A1 is part of the double-hit expression signature (HGBL-DH/TH) in diffuse large B-cell lymphoma (DLBCL) (Ennishi et al. 2019), is more expressed in germinal center B-cell (GCB) DLBCL where it sustains lymphoma cells’ growth (Sartori et al, 2022). Here, we quantified the ORs expression in lymphoma clinical specimens and performed functional experiments in cell lines. Methods. Gene expression data for clinical samples were obtained from the public dataset phs001444.v2.p1. Patients were stratified according to OR expression groups. Kaplan–Meier survival analysis was performed to evaluate differences in outcome between groups, and LIMMA was used to identify OR-associated gene expression signatures. OR’s expression was first evaluated using a previously generated total RNA-Seq dataset from 47 B-cell lymphoma cell lines and then was assessed by qPCR across five human cell lines: two GCB-DLBCL (OCI-Ly7 and VAL), two activated B-cell-like (ABC)-DLBCL (U2932 and HBL1), and one mantle cell lymphoma (MCL) (JEKO1). OR2B6 silencing was performed in HBL1 and JEKO1 cells using doxycycline-inducible shRNAs, and viability was monitored via live imaging. Results. OR13A1, OR2B6, and OR51E2 were expressed in at least 50% of patients, but only OR13A1 and OR2B6 were detected in RNASeq data from lymphoma cell lines. The lack of OR51E2 in cell lines was suggestive of its expression by cells of the tumor microenvironment (TME), such as macrophages, as recently reported in prostate cancer (Marelli et al, 2025). As expected, OR13A1 was associated with HGBL (DH/TH)-related genes and germinal center B cell-like signatures. OR2B6 was associated with ABC-DLBCL genes and down-regulated HGBL (DH/TH) genes. By qPCR OR13A1 was predominantly expressed in GCB-DLBCL (2-ΔCt = 0.015), moderately in MCL (2-ΔCt = 0.007) and almost absent in ABC-DLBCL (2-ΔCt < 0.0004). In contrast, OR2B6 was mostly expressed in ABC-DLBCL (2-ΔCt = 0.004) and MCL (2-ΔCt = 0.002), with negligible expression in GCB-DLBCL (2-ΔCt < 0.0004). While OR13A1 had already been identified in our lab as an essential gene in GCB-DLBCL, OR2B6 was validated as essential in HBL1 and JEKO1 cell lines derived from ABC and MCL, respectively. Conclusion. These findings suggest that three chemosensors from the OR family may play a role in lymphoma development: OR13A1 (GCB-DLBCL), OR2B6 (ABC-DLBCL and MCL), expressed by lymphoma cells, and OR51E2, expressed by the TME. Further research is needed to identify their ligands, understand their biological mechanisms, and evaluate their therapeutic potential. Citation Format: Giulio Sartori, Sara Sabatini, Alessandro Ghiringhelli, Natalie Amberg, Luciano Cascione, Jacopo Sgrignani, Hiroaki Matsunami, Andrea Cavalli, Francesco Bertoni. OR2B6 is a chemosensor associated with activated B-cell like diffuse large B cell and mantle cell lymphoma [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 A014.

Background The Pfizer-BioNTech coronavirus vaccine (BNT162b2) was among the first nanoparticle-based vaccines approved by the World Health Organisation (WHO) and demonstrated 95% efficacy against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Despite its success, the precise immune mechanism behind its effectiveness remains poorly understood. This study investigated the early immune responses occurring in the draining lymph node (dLN) following vaccination. Methods A well-established murine vaccination model was used to investigate the distribution and immunological effects of BNT162b2 in vivo. Vaccine trafficking to the dLN, cellular uptake, and spike protein expression were assessed following immunisation. Single-cell transcriptomic analyses and functional in vivo experiments were performed to identify the immune cell populations involved in orchestrating vaccine-induced responses and to characterise their interactions with adaptive immune cells. Findings BNT162b2 was rapidly transported to the dLN, where it was predominantly captured by leukocytes that subsequently expressed the SARS-CoV-2 spike protein. Among these cells, plasmacytoid dendritic cells (pDCs) emerged as regulators involved in both inflammatory and humoural immune responses. Single-cell transcriptomic profiling revealed active interactions between pDCs and CD8+ T cells. Functional in vivo studies further demonstrated that pDCs promoted CD8+ T-cell activation and expansion, indicating an involvement in shaping adaptive immunity following vaccination. Interpretation These findings identify pDCs as antigen-presenting cells involved in coordinating the immune response to BNT162b2. By regulating both innate and adaptive immune pathways and facilitating CD8+ T-cell activation, pDCs appear to play a functional role in the efficacy of this mRNA vaccine. This study provides additional insights into the immunological processes underlying mRNA vaccine-induced protection and may inform the future design and optimisation of mRNA-based immunotherapies and vaccines. Funding Swiss National Science Foundation, Leonardo Foundation.

Supplementary Fig. S5. Kaplan–Meier survival analysis in (A) Ramos and (B) WSU-DLCL2 xenograft models. Kaplan–Meier survival plots showing percentage animal survival (A) over 60 days in Ramos xenograft; and (B) over 59 days in WSU-DLCL2 cancer xenograft. QD once daily.

Abstract Background. BM011 is the first-in-class WASP (Wiskott-Aldrich Syndrome Protein) activator (Spriano et al, 2024). WASP regulates cytoskeletal remodeling, immune synapse formation, vesicle trafficking, and mechano-metabolic coupling. Aberrant actin dynamics and rewiring of the WASP pathway contribute to proliferation, metabolic flexibility, and stress resistance in lymphomas and other cancers. Here, we investigated the mechanism of action of BM011 by combining transcriptomics, proteomics, and a genome-wide CRISPR-Cas9 knockout screen in lymphoma cells treated with the compound. Methods. JEKO1 mantle cell lymphoma cells were treated with BM011 for 8h and analyzed by RNA-Seq and mass spectrometry (MS). A genome-wide CRISPR-Cas9 knockout screen was conducted following 14-day exposure to BM011. Results. Proteomics revealed a strong downregulation of mitochondrial import and respiratory chain proteins in BM011-treated cells, while many RNAs coding for the same molecules were upregulated, consistent with an attempt to compensate for mitochondrial stress. Similarly, ribosomal proteins decreased, while ribosomal transcripts increased, suggesting a proteotoxic stress response and a translational offset. Stress-response genes (e.g., HSPA family), cytoskeleton, and apoptotic pathways were strongly induced, indicating proteotoxic pressure, cytoskeleton remodeling, and apoptosis induction following cytoskeletal forcing. At the genetic screen, the loss of many mitochondrial respiratory chain genes sensitized cells to BM011, confirming their importance in mitigating BM011-induced mitochondrial collapse. In contrast, the knockout of FUNDC2 and TIMM23, not part of the mitochondrial respiratory chain, conferred resistance, likely due to the interruption of mitochondrial quality control and decreased apoptosis upon BM011 treatment. The genetic screen also revealed a divergent vulnerability to glycolysis. Knockout of genes coding for enzymes of the upper glycolytic process (HK2, PFKM, PFKL, GPI, PFKFB3) conferred resistance, while knockout of genes coding for proteins in the lower part of glycolysis (PGK1, ENO1, PKM, PFKFB4) increased sensitivity. This pointed to aldolase and the fructose-1,6-bisphosphate node as a critical metabolic point. Aldolase can indeed bind to F-actin and WASP, inhibiting actin polymerization. Additionally, an excess of fructose-1,6-bisphosphate (F16bP) inhibits aldolase binding to F-actin. We proved that F16bP supplementation or aldolase inhibition increased the sensitivity of BM011 in otherwise resistant lymphoma cells. Conclusion. BM011-driven activation of WASP induces cytoskeletal stress, mitochondrial import failure, and a proteotoxic response, with upper-glycolysis and aldolase acting as a metabolic safety valve. These reveal druggable metabolic-cytoskeletal vulnerabilities in lymphoma. Citation Format: Filippo Spriano, Luciano Cascione, Stephen Croke, Digvijay Gahtory, Maurits van den Nieuwboer, Francesco Bertoni. WASP activation by BM011 triggers mitochondrial collapse and aldolase-dependent glycolytic vulnerability in 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 4512.

Numerous studies have described the altered expression and the causal role of microRNAs (miRNAs) in human cancer. However, to date, efforts to modulate miRNA levels for therapeutic purposes have been challenging to implement. Here we find that nucleolin (NCL), a major nucleolar protein, posttranscriptionally regulates the expression of a specific subset of miRNAs, including miR-21, miR-221, miR-222, and miR-103, that are causally involved in breast cancer initiation, progression, and drug resistance. We also show that NCL is commonly overexpressed in human breast tumors and that its expression correlates with that of NCL-dependent miRNAs. Finally, inhibition of NCL using guanosine-rich aptamers reduces the levels of NCL-dependent miRNAs and their target genes, thus reducing breast cancer cell aggressiveness both in vitro and in vivo. These findings illuminate a path to novel therapeutic approaches based on NCL-targeting aptamers for the modulation of miRNA expression in the treatment of breast cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical regulatory roles in gene expression by targeting messenger RNAs (mRNAs) for degradation or translational inhibition. Their ability to modulate multiple genes simultaneously places them at the center of many biological processes, including those involved in tumorigenesis. Depending on their targets, miRNAs can act as oncogenes or tumor suppressors, contributing to cancer development and progression. Advances in high-throughput sequencing now allow simultaneous profiling of miRNA and mRNA expression in the same biological samples. When properly integrated, these datasets can uncover functional miRNA-mRNA regulatory networks, reveal cancer-specific signatures, and identify novel biomarkers or therapeutic targets. However, the integrative analysis of miRNA and mRNA expression data remains computationally challenging and requires well-structured pipelines and rigorous statistical approaches. In this chapter, we present updated computational strategies to identify biologically relevant miRNA-mRNA interactions through expression correlation, target prediction, and functional enrichment. The chapter includes step-by-step protocols, example R code, and recommendations for integrating additional data layers such as copy number variation and DNA methylation, with the goal of improving the robustness and interpretability of miRNA-based analyses in cancer.

Abstract Background: BCL2 inhibitors (i) and BTK-directed agents, including BTK degraders (d), are essential components of modern therapy for B-cell lymphomas. However, acquired resistance often limits long-term benefits. Identifying the molecular mechanisms that underlie resistance and understanding reciprocal drug sensitivities may help guide the rational sequencing of treatments. Here, we investigated the mechanisms of secondary resistance to the BCL2-i venetoclax and the BTK-d bexobrutideg (NX-5948) in models of marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL). Methods: Derivatives of Karpas1718 (MZL) and REC1 (MCL) with acquired resistance were developed through chronic exposure to increasing doses of venetoclax or bexobrutideg. Parental and resistant lines were characterized using drug sensitivity assays, apoptosis and cell cycle analyses, molecular profiling, and immunoblotting of apoptotic regulators and BCR signaling components. Results: Venetoclax-resistant models from both lymphoma subtypes exhibited broad cross-resistance to other BCL2-i and upregulation of BCL-xL, with MZL cells also resistant to MCL1-i. Regulation of anti-apoptotic proteins varied among models: BCL-xL increased in both venetoclax-resistant models, whereas MCL1 increased only in Karpas1718. All venetoclax-resistant cell lines retained sensitivity to BTK-d, revealing a potential therapeutic vulnerability. Resistance to BTK-d was highly model-specific. In Karpas1718-resistant cells, no BTK degradation occurred after bexobrutideg exposure, and these cells were also resistant to BGB-16673, another BTK-d. Nonetheless, they remained sensitive to BTK-i and all BCL2-i, suggesting that switching to a different drug class could restore their response. Conversely, REC1 derivatives became resistant to both BTK-d and BTK-i. Since resistance was linked to continued drug-induced BTK degradation, these findings indicate a BTK-independent resistance mechanism. Interestingly, the bexobrutideg-resistant REC1 cells were also resistant to BCL2-i. No mutations were observed in BTK, PLCG2, or BCL2. Conclusions: In two distinct B-cell lymphoma models, resistance to venetoclax and BTK-d developed through target-specific and model-specific adaptations involving anti-apoptotic reprogramming, altered BCR signaling dependence, and incomplete BTK degradation. Notably, cross-drug sensitivities persisted in several contexts: venetoclax-resistant MZL and MCL cells remained susceptible to BTK degradation, while BTK-d-resistant MZL cells continued to respond to BCL2 inhibition. These insights emphasize clinically actionable strategies for treatment sequencing and support precision approaches to overcome therapeutic resistance in B-cell lymphoma. Citation Format: Alberto J. Arribas, Camilla Scalise, Eleonora Cannas, Maria Elena Carazzolo, Luciano Cascione, Andrea Rinaldi, Carlo Visco, Davide Rossi, Francesco Bertoni. Mechanisms of acquired resistance to BTK and BCL2 inhibitors reveal clinically actionable vulnerabilities in B-cell 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 1857.

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.

Supplementary Fig. S2. The in vitro antitumor activity of ADCT-602 does not correlate with CD22 levels in B-cell lymphomas. This figure shows a correlation of ADCT-602 IC50 values versus (A) cell surface protein expression via absolute fluorescence quantitation with Quantum Simply Cellular microspheres, (B) CD22 RNA expression levels measured via Illumina HT-12 microarrays, and (C) targeted RNA-Seq with the HTG EdgeSeq Oncology Biomarker Panel. On each plot, the x-axes are the ADCT-602 IC50 values presented as log2, and the y-axes are CD22 expression levels presented as log2. Cell lines derived from Hodgkin and T-cell lymphomas have been excluded. IC50 50% inhibitory concentration.

Supplementary Fig. S4. Time course of DNA ICL formation in KARPAS-299 cells following a 2-hour treatment with ADCT-602 or SG3199. Results are presented as a mean percentage decrease in OTM ± SD (n = 4). ICL interstrand crosslinking; OTM Olive tail moment.

Background: CXCR4 is a chemokine receptor implicated in B-cell lymphoma pathogenesis and therapeutic resistance. Targeting CXCR4 is under active investigation using small molecules, antibodies, and peptide antagonists. We evaluated the antitumor activity of the synthetic CXCR4 inhibitors SPX5551 and balixafortide, alone and in combination, across B-cell lymphoma models.Methods: Binding modes were analyzed by molecular docking and molecular dynamics simulations. In vitro assays assessed single-agent and combinatorial effects with BTK inhibitors, PI3K inhibitors, rituximab, and R-CHOP in 20 lymphoma cell lines, including models with acquired resistance. Primary chronic lymphocytic leukemia (CLL) samples were used for translational validation. Transcriptomic and signaling analyses investigated mechanisms of synergy.Results: Structural modeling revealed highly similar CXCR4 binding modes for SPX5551 and balixafortide. SPX5551 showed limited single-agent activity but restored sensitivity to BTK and PI3K inhibitors in resistant marginal zone lymphoma models. Across mantle cell lymphoma (MCL), CLL, and diffuse large B-cell lymphoma models, SPX5551 enhanced the efficacy and/or potency of ibrutinib, copanlisib, rituximab, and R-CHOP. In MCL, dual CXCR4 and BTK inhibition synergistically induced apoptosis, suppressed NF-κB, AKT, and ERK signaling, and repressed inflammatory and MYC-driven transcriptional programs. These effects were confirmed in primary CLL samples, where combined treatment increased cytotoxicity and more effectively inhibited survival signaling than single agents.Conclusions: Although CXCR4 inhibition alone has limited cytotoxicity, it potentiates targeted agents and chemo-immunotherapy, including in drug-resistant models and primary CLL cells. These findings support clinical evaluation of CXCR4 blockade as a combinatorial strategy in B-cell malignancies.

Diffuse large B-cell lymphoma (DLBCL) remains a challenging disease with limited therapeutic options beyond standard immunochemotherapy. ETS transcription factors, including SPIB and SPI1, are implicated in lymphoma pathogenesis and can be targeted by the small molecule TK216, which disrupts ETS-DHX9 interactions. To explore mechanisms of resistance, we generated stable TK216-resistant clones from the ABC-DLBCL line U2932. Resistant clones exhibited a 4-5-fold increase in IC50 values and lost the ability to undergo G2-M arrest upon treatment. Transcriptomic and mutational analyses revealed three resistance patterns: (i) MDR1/ABCB1 overexpression, leading to multidrug efflux; (ii) Cluster A, enriched for proliferation, Wnt, and transcriptional programs, with mutations in ESR2, USP24, and SFSWAP; and (iii) Cluster B, characterized by actin/microtubule remodeling, altered metabolism, and mutations in SRSF11 and PATJ. Pharmacologic screening revealed an increased sensitivity of resistant cells to BCL2, MCL1, and XPO1 inhibitors, while also showing reduced sensitivity to aurora kinase and microtubule-targeting agents. Venetoclax and selinexor retained activity in resistant models, supporting their potential for rational combinations with TK216. These findings demonstrate that multiple, heterogeneous mechanisms drive resistance to ETS inhibition in DLBCL, highlighting therapeutic strategies to overcome it.

Cyclin-dependent kinase 9 (CDK9) drives transcriptional elongation and supports the expression of short-lived oncogenic and anti-apoptotic proteins such as MYC and MCL1. PRT2527 is a potent, selective CDK9 inhibitor currently in early clinical development. We evaluated its preclinical activity in marginal zone lymphoma (MZL) models, including cell lines with acquired resistance to BTK, PI3K, and BCL2 inhibitors. Short exposure (4 hours) to PRT2527 produced nanomolar cytotoxicity across all tested MZL cell lines, with efficacy maintained in resistant derivatives. Transcriptomic profiling of VL51 cells showed broad gene repression, including MYC, IRF4, NF-κB-related genes, and MCL1, alongside increased expression of HLA class II genes. Moreover, comparison with additional CDK inhibitors revealed a similar transcriptional repression signature, underscoring a conserved CDK-dependent regulatory network. Protein analyses confirmed rapid depletion of MCL1, MYC, RNA polymerase II, and IRF4. Flow cytometry validated increased HLA class II and decreased HLA class I surface expression. Combination studies demonstrated additive to synergistic effects with BTK inhibition (ibrutinib) or dual PI3K/BCL2 inhibition (copanlisib plus venetoclax), independent of baseline drug sensitivity. Mechanistically, these combinations may enhance apoptosis by concurrently suppressing survival signaling and transcriptional addiction. Analysis of patient samples revealed high CDK9 expression, further supporting the biological relevance and therapeutic rationale for targeting CDK9 in this disease. Our findings support the development of CDK9-based combination strategies for relapsed/refractory MZL and other B-cell malignancies, with an additional potential for integration with immunotherapies. Key Points 1. CDK9 inhibitor PRT2527 kills marginal zone lymphoma cells, regardless of whether they are resistant to other targeted drugs. 2. PRT2527 boosts the effects of BTK, PI3K, and BCL2 inhibitors and alters immune-related gene expression. ![Figure][1] ### Competing Interest Statement Potential Competing Interests Alberto J. Arribas: travel grant from AstraZeneca and Floratek Pharma, advisory board fee from PentixaPharm. Luciano Cascione: institutional research funds from Orion; travel grant from HTG. Davide Rossi: grant support from Gilead, AbbVie, Janssen; honoraria from Gilead, AbbVie Janssen, Roche; scientific advisory board fees from Gilead, AbbVie, Janssen, AstraZeneca, MSD. Anastasios Stathis: institutional research funds from Pfizer, MSD; Roche, Novartis, Amgen, Abbvie, Bayer, ADC Therapeutics, MEI Therapeutics, Philogen, Celestia. Astra Zeneca; travel grant from AbbVie and PharmaMar; consulting fee paid to institution from Jansen, Roche, Eli Lilly. Diane Heiser: Prelude employment Francesco Bertoni: institutional research funds from ADC Therapeutics, Bayer AG, BeiGene, Floratek Pharma, Helsinn, HTG Molecular Diagnostics, Ideogen AG, Idorsia Pharmaceuticals Ltd., Immagene, ImmunoGen, iOnctura, Mabtree, Menarini Ricerche, Nordic Nanovector ASA, Oncternal Therapeutics, Spexis AG; consultancy fee from BIMINI Biotech, Floratek Pharma, Helsinn, Immagene, Menarini, Vrise Therapeutics; advisory board fees to institution from Novartis; travel grants from Amgen, Astra Zeneca, iOnctura. The other Authors have nothing to disclose. Swiss National Science Foundation, https://ror.org/00yjd3n13, SNSF 31003A_163232/1 Swiss Cancer Research, KFS-4727-02-2019 Prelude [1]: pending:yes

Supplementary Fig. S8. Quantification of cynomolgus monkey CD3+, platelets, reticulocytes, and neutrophils after dosing ADCT-602 at 0.6 or 0.9 mg/kg (2 doses, 21 days apart). Data presented as mean ± SEM (each group, n = 3). SEM standard error of mean.

Supplementary Fig. S7. Cynomolgus monkey body weight after dosing ADCT-602 at 0.6 or 0.9 mg/kg (2 doses, 21 days apart). Data presented as mean body weight (kg) ± SEM (each group, n = 3). SEM standard error of mean.

Background: This phase I trial aimed to assess the safety and preliminary activity of the combination of copanlisib with venetoclax in previously treated patients with B-cell non-Hodgkin lymphoma, excluding mantle-cell lymphoma patients. Methods: Intravenous copanlisib on days 1, 8, and 15 and oral venetoclax once daily (starting on day 2 of cycle 1, continuously) were administered in 28-day cycles up to a maximum of 12 cycles. Starting doses were 600 mg of venetoclax and 60 mg of copanlisib. Results: A total of seven patients were enrolled. The first two presented dose-limiting toxicities, five additional patients were treated at a lower dose level (45 mg of copanlisib and 400 mg of venetoclax), with no dose-limiting toxicities observed among three evaluable patients. The most frequent treatment-related adverse event of grade 3 or higher was neutropenia (n = 3), followed by thrombocytopenia (n = 2) and hypertension (n = 2). Four treatment-related serious adverse events occurred, including grade 3 febrile neutropenia in one patient and respiratory infections in three patients. Two patients had complete responses, and two had partial responses. Transcriptomic analysis of lymphoma samples showed enrichment of genes coding for proteins involved in B-cell receptor signaling in responding patients. The study was terminated prematurely due to a decision by the pharmaceutical company. Conclusions: Due to the limited sample size, no definite conclusions regarding safety or activity of the combination can be drawn. The two drugs could not be combined at their respective single-agent doses. Responses were observed in heavily pretreated patients. Transcriptomic analyses suggested a possible association between response and B-cell receptor signaling, which warrants further investigation.

Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease, with the activated B-cell-like (ABC) subtype showing inferior outcomes. The ETS transcription factors ETS1 and FLI1 are recurrently gained and functionally relevant in DLBCL, yet their pathogenic role remains to be fully elucidated. Here, we describe their cooperation with the RNA regulatory machinery, demonstrating that the RNA helicase DDX21 is a central effector of the ETS1/FLI1 transcriptional network in ABC-DLBCL. Our proteomic analyses revealed that ETS1 physically interacted with DDX21 and other RNA processing factors. As ETS1, DDX21 was preferentially expressed in ABC-DLBCL, particularly in the MCD/C5 genetic subtype, and it was associated with adverse clinical outcomes in this lymphoma subtype. Genetic and pharmacological studies demonstrated that DDX21 was essential for ABC-DLBCL cell proliferation. DDX21 also coordinated various transcriptional programs, which were revealed by integrated RNA-Seq, small RNA-Seq, ChIP-Seq, and Capture Hi-C analyses. DDX21-dependent transcription was relevant for ribosome biogenesis, MYC signaling, cell cycle progression, and immune evasion, but also regulated non-coding RNA networks, including microRNAs and small nucleolar RNAs, in particular SNORA37. Collectively, our data establish DDX21 as a nucleolar hub linking ETS transcription factors to coding and non-coding RNA programs that sustain aggressive lymphoma biology. These findings suggest DDX21 and ETS-RNA helicase complexes as promising therapeutic vulnerabilities in ABC-DLBCL.