Data collection and refinement statistics of the dCK/UDP/EdC crystal structure.

Mechanism of action of compounds identified in PRISM screen as top hits with similar efficacy and sensitivity profile as EdC.

ADME and PK properties of 1µM EdC in vitro.

Individual blood concentrations (ng/mL) and pharmacokinetic parameters for EdC after IP injection.

Additional EdC metabolism data.

Supplementary Figure Legends

Schematic of semi-unbiased screening approach.

Sequential steps and intermediates for synthesis of EdC (compound 13).

Abstract Aggressive B-cell lymphomas have significantly high relapse/refractory rates of 20-40%, and outcomes in these patients are very poor, warranting an urgent need for novel, more targeted therapeutic strategies. Using a combination of biological and biochemical approaches, we identified a previously unknown, targetable vulnerability in diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma, including those with genetic alterations that lead to more aggressive and challenging to treat disease, such as p53 mutations and MYC overexpression with/without BCL2 overexpression. With a new bispecific compound, we target two oncoproteins, both of which are overexpressed in B-cell lymphomas, and observed high efficacy with apoptosis induction. The targeted bispecific compound binds to two oncoproteins and promotes their degradation, leading to activation of the p53 family member, p73, in p53 mutant lymphomas and apoptosis of cell lines and patient samples. Moreover, loss of the oncoproteins with the bispecific compound in vivo effectively reduces tumor volumes and significantly extends survival of mice with DLBCL and Burkitt lymphoma xenografts. We further evaluated the mechanism by which the bispecific compound promotes p73-mediated lymphoma cell death. Prior to apoptosis, caspase-independent DNA double-strand breaks and DNA damage signaling, the stimuli for p73 activation and apoptosis, was observed. Our data underscore dual targeting of two oncoproteins as a novel vulnerability with treatment potential for aggressive B-cell lymphomas. Our results also yield important insights into DNA instability in rapidly growing lymphomas that could be further exploited. Citation Format: Brittany M. Mahon, Clare M . Adams, Marco Boccitto, Jessica Cammarota, Ramkrishna Mitra, Christine M. Eischen. Amplifying B-cell lymphoma apoptosis by targeting two oncoproteins with a new bispecific compound [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 A036.

RNA-seq analysis of T-ALL Jurkat and DLBCL SUDHL-10 after 16-hour treatment with 1µM EdC.

Evaluation of in vitro ADME properties and in vivo pharmacokinetics of EdC.

Mutation-based sensitivity patterns and correlation analysis of CRISPR knock-out with EdC sensitivity.

EdC sensitivity correlates with sensitivity to replication stress-inducing compounds.

Additional methods for PRISM screen and EdC synthesis.

Supplementary Materials and Methods, Tables S1-S3, Figures S1-S7

Abstract: The consequences of activated innate immune signaling in acute myeloid leukemia (AML) is not well understood. Using ligands directed at the toll-like family receptors (TLR) in models of high-risk AML, we uncover that TLR2 ligands exert unique antileukemic effects that are distinct from other TLRs. Although TLR2 signaling broadly induces inflammatory gene expression in AML cells, at the single-cell level, cell-type–dependent, divergent transcriptional responses coordinate cellular outputs of proliferation, differentiation, cell death, and activation of immune cell function. TLR2 ligands were the only TLR agonists capable of extending survival of AML-bearing mice through leukemia stem cell (LSC) reprogramming that elevated major histocompatibility complex (MHC) class II surface expression and ultimately impaired self-renewal. We find that the coexpression of TLR2 and MHCII genes is associated with better overall survival in patients with AML, which is consistent with our observations of activated TLR2 signaling in mice. These data reveal functional TLR2 signaling critically antagonizes leukemogenesis and emphasizes a role for TLR2 agonism in AML.

AbstractAnticancer nucleosides are effective against solid tumors and hematologic malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4′-ethynyl-2′-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å cocrystal structure of DCK bound to EdC and UDP reveals how the rigid 4′-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared with FDA-approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a preclinical nucleoside prodrug candidate for DLBCL and ALL.

Aberrant inflammatory signaling is a hallmark of myeloid malignancies and is generally thought to promote disease. Few studies have systematically dissected the molecular and cellular mechanisms of pathogen-induced inflammation in myeloid disease settings. Using our previously established immune-competent mouse model of DNTM3A/FLT3-mutant acute myeloid leukemia (FD AML), we screened a panel of synthetic Toll-like receptor (TLR) agonists to mimic pathogen-specific responses. Activation of TLR2 heterodimers, but not other TLRs, significantly extended overall survival of leukemia bearing mice. Moreover, TLR2 is the highest expressed TLR in AML patients regardless of mutation profile. These data led us to explore the impact of TLR2 mediated inflammatory signaling on AML disease processes. To understand AML-cell intrinsic versus extrinsic effects, we conducted a series of reciprocal syngeneic transplants using FD AML and Tlr2-/-FD AML into wild-type (WT) or Tlr2-/- recipients. Note, Tlr2 deletion does not significantly alter the development or progression of FD AML. After AML establishment, mice were treated with a single dose of Tlr1/2 specific agonist, Pam3CSK4. This revealed Tlr2 expression on AML cells is essential for significantly prolonged survival with Pam3CSK4 treatment. Given that TLRs have direct and indirect signaling with functional phenotypic consequences, we employed single-cell RNA sequencing coupled with cell surface proteins (CITE-seq) to unbiasedly investigate the pleiotropic effects of Tlr2 activation. We identified 21 clusters that recapitulate the hierarchical nature of FD AML. Pam3CSK4 significantly decreased the fraction of AML cells in stem/progenitor clusters, expanded AML-derived monocyte precursor clusters, and gave rise to a new AML-derived macrophage cluster. There were also decreased AML-derived mature neutrophil clusters despite expansion of neutrophil precursor clusters. We validated these AML cell states using the reciprocal Tlr2 proficient/deficient transplantation models. Our data indicate that Tlr2 activation drove effector macrophages (CD11b+F4/80+ MHCII+ CD80+/86+) with elevated phagocytic activity from AML. In contrast, Pam3CSK4 blocked maturation of AML-derived neutrophils in an AML-non-autonomous manner. Cytokine analyses showed G-CSF production downstream of Tlr2 activation in the microenvironment prevented the cell intrinsic Tlr2-mediated neutrophil maturation of AML cells. By CITE-seq, Cepbewas significantly reduced in AML-derived neutrophil precursors, indicating the block by G-CSF may be happening at a precursor stage. Indeed, FD AML co-treated with anti-G-CSF neutralizing antibodies and Pam3CSK4 restored AML-derived neutrophil differentiation, but at the expense of monocyte differentiation, through a bipotential intermediate. Lastly, to understand whether Tlr2 activation on AML cells causes a transient reduction in stem/progenitor numbers or a sustained functional impairment of AML stem cell activity, we transplanted limiting numbers of FD AML stem/progenitor cells from Pam3CSK4 and vehicle treated mice into WT recipients. We observed a 5-fold decrease in leukemia initiating cell activity with Pam3CSK4 treated donors. In summary, we discovered Tlr2 activation induces pleiotropic effects on a variety of cell types in AML, ultimately impacting overall survival. Our results shed light on the functional consequences of pathogen recognition by AML cells that may be leveraged for novel treatments of this deadly disease.

Clinical trials with single-agent venetoclax/ABT-199 (anti-apoptotic BCL2 inhibitor) revealed that diffuse large B-cell lymphoma (DLBCL) is not solely dependent on BCL2 for survival. Gaining insight into pathways/proteins that increase venetoclax sensitivity or unique vulnerabilities in venetoclax-resistant DLBCL would provide new potential treatment avenues. Therefore, we generated acquired venetoclax-resistant DLBCL cells and evaluated these together with intrinsically venetoclax-resistant and -sensitive DLBCL lines. We identified resistance mechanisms, including alterations in BCL2 family members that differed between intrinsic and acquired venetoclax resistance and increased dependencies on specific pathways. Although combination treatments with BCL2 family member inhibitors may overcome venetoclax resistance, RNA-sequencing and drug/compound screens revealed that venetoclax-resistant DLBCL cells, including those with TP53 mutation, had a preferential dependency on oxidative phosphorylation. Mitochondrial electron transport chain complex I inhibition induced venetoclax-resistant, but not venetoclax-sensitive, DLBCL cell death. Inhibition of IDH2 (mitochondrial redox regulator) synergistically overcame venetoclax resistance. Additionally, both acquired and intrinsic venetoclax-resistant DLBCL cells were similarly sensitive to inhibitors of transcription, B-cell receptor signaling, and class I histone deacetylases. These approaches were also effective in DLBCL, follicular, and marginal zone lymphoma patient samples. Our results reveal there are multiple ways to circumvent or overcome the diverse venetoclax resistance mechanisms in DLBCL and other B-cell lymphomas and identify critical targetable pathways for future clinical investigations.