Lactate-Mediated Histone H3K9 Lactylation Facilitates Tumorigenesis of T-Cell Lymphoma Via Activation of SFXN1 Expression

Gli1 Inhibitor GANT61 Exhibits Antitumor Efficacy in T-Cell Lymphoma Cells through Down-Regulation of p-STAT3 and SOCS3 Pathways

Darinaparsin in T-Cell Lymphoma (TCL) and Hodgkin Lymphoma (HL) Cells Lines and SCID Xenograft Mouse Models: Single-Agent Activity and Synergistic Cell Death Combined with a PI3K/mTOR Inhibitor

Histone Deacetylase Inhibitors Downregulate CCR4 Expression and Decrease Mogamulizumab Efficacy in CCR4-Positive Mature T-Cell Lymphomas

Introduction: Peripheral and cutaneous T cell lymphomas (TCL) are highly heterogeneous diseases that in the relapsed/refractory (R/R) stage have poor outcome. JAK/STAT, Pi3K/AKT and MAP kinase signaling are commonly involved in the pathogenesis of these lymphomas, and the efficacy of small molecule inhibitors targeting these pathways is being assessed in phase II trials. However, biomarkers to define patients most likely to benefit from specific inhibitors are lacking and mechanisms of acquired resistance in TCLs have not been reported. Methods: We are conducting two early phase trials in R/R TCL: 1) NCT02974647 targets JAK1/JAK2 with ruxolitinib (RUX, a JAK 1-2 inhibitor) and has enrolled 53 patients and 2) NCT02783625 targets Pi3K/AKT with duvelisib (DUV, a Pi3K gamma-delta inhibitor) in combination with either romidepsin or bortezomib and has enrolled 92 patients. We analyzed pre-, on and post- treatment biopsies with multiplex immunofluorescence (mIF) using the Vectra platform, and analyzed images for marker expression and cellular location using HALO software. We designed multiple mIF panels with 6 markers per slide, 3 identifying TCL cells based on disease-specific phenotypes (e.g. CD4+PD1+CD8- for angioimmunoblastic TCL), pSTAT3,5, pS6, and a macrophage marker (CD68). Areas of TCL involvement within biopsy slides were manually defined during image analysis based on mIF and comparison to clinical immunohistochemistry. Differences in marker expressions were analyzed with the Wilcoxon test using the program R. Results: We analyzed 53 biopsies from 39 patients with high-quality mIF images, of which 17 were from lymph nodes and 36 were from extranodal sites. Of 39 patients, 13 were treated with RUX (9 biopsies pre-, 7 on, 4 post- treatment) and 26 with DUV (20 biopsies pre-, 6 on, 10 post- treatment). Median number of total TCL cells in pre-treatment biopsies was 1041 (range, 97-6463) and median TCL cell fraction within involved areas was 27.86% (range, 5.12-88.02%). Median macrophage fraction within involved areas was 5.92% (range, 2.89-11.88%). For either agent, response to treatment was not associated with: 1) quantity of macrophages (p=0.1 for responders vs non-responders to DUV, p=0.53 for RUX), 2) average distance between TCL cells and closest macrophage, 3) TCL cell fraction within involved areas, or 4) fraction of TCL cells expressing pSTAT3/5 (Figure). In contrast, pS6 expression in <25% of TCL cells within pre-treatment biopsy completely distinguished responders from non-responders (Figure; p=0.02). Neither agent induced consistent changes in expression of pSTAT3/5 or pS6 at the on or post-treatment timepoints. Conclusions: mIF is a feasible platform for biomarker discovery and translation in TCL, a set of lymphomas with heterogeneous immunophenotypes. pS6 expression in <25% of TCL cells may predict response to ruxolitinib, suggesting that activation of S6 kinase downstream of PI3K or MAP kinase, may overcome dependence on JAK/STAT signaling. This is also the first data suggesting that ruxolitinib responses involve a TCL cell-intrinsic mechanism of action. Based on these data, we are assessing the use of pS6 IHC as a clinically-validated biomarker in patients with TCL prior to ruxolitinib. Citation Format: Paola Ghione, Alison Moskowitz, Priadarshini Kumar, Andrea Knezevic, Venkatraman Seshan, Eric Jacobsen, Jia Ruan, Johnathan Schatz, Sarah Noor, Patricia Myskowski, Helen Hancock, Theresa Davey, Obadi Obadi, Carlissa Onwasigwe, Nivetha Ganesan, Lauren Pomerantz, Christine Jarjies, Allison Sigler, Mark Geyer, Ariela Noy, David Straus, Natasha Galasso, Giorgio Inghirami, Steven Horwitz, David Weinstock, Travis Hollmann, Ahmet Dogan. Multispectral immunofluorescence identifies pS6 as a biomarker of intrinsic resistance to ruxolitinib in patients with relapsed/refractory T-cell lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4248.

Inhibition of IL2-Inducible T-Cell Kinase (ITK)-Mediated Chemoresistance By Ibrutinib in T-Cell Lymphoproliferative Disorders

Targeting B- and T-cell lymphomas with anti-CXCR5 CAR T cell therapy

T-cell lymphoma (TCL) is a group of non-Hodgkin's lymphoma with high heterogeneity and unfavorable prognosis. Current standard treatments have demonstrated limited efficacy in improving the outcomes for TCL patients. Therefore, identification of novel drug targets is urgently needed to improve the prognosis of TCL patients. Through multi-omics analysis, aberrant expression of threonine tyrosine kinase (TTK) in TCL is identified. High expression of TTK is closely associated with poor prognosis in TCL patients. Targeting TTK through gene knockdown exerts anti-tumor effects in vitro and in vivo, including inhibiting the cell proliferation, inducing G2/M phase arrest, enhancing DNA damage and cell apoptosis. Mechanically, p38α is identified as the potential phosphorylation substrate of TTK through phosphoproteomic quantification and motif prediction. Furthermore, inhibition of TTK suppresses activation of p38α through dephosphorylating it at Thr180/Tyr182, thereby promoting the activation of AMPK/mTOR pathway. In addition, targeting TTK enhances the autophagy in TCL cells through dephosphorylating p38α. CFI-402257, a specific inhibitor of TTK, is found to exhibit anti-tumor effects and exerted synergistic efficacy with PI3K inhibitor, Duvelisib, in TCL. The study shows that TTK contributes to the development of TCL by regulating p38α-mediated AMPK/mTOR pathway. CFI-402257 is expected to be a promising strategy for TCL treatment.

The 2nd Generation Proteasome Inhibitor, MLN2238: Potent Induction of Cell Death in T-Cell Lymphoma (TCL) and Hodgkin Lymphoma (HL) Cell Lines and in Two Human Lymphoma Xenograft Models.

Tissue regeneration relies on precise molecular mechanisms controlling cell-fate transitions, with metabolism emerging as a key regulator. Lactate-derived histone lactylation has recently been identified as an epigenetic modification regulating gene expression across various biological processes. Here, we report an increase in global histone lactylation in the mesenchyme and osteoblasts of the zebrafish caudal fin during early regeneration. Our findings demonstrate that this epigenetic modification is functionally regulated by increased lactate levels, while the inhibition of glycolysis and lactate production significantly reduces histone lactylation. Transcriptomic profiling under reduced lactylation revealed the downregulation of proliferative and chromatin-remodeling programs. This suggests a model in which injury-induced, lactate-driven histone lactylation sustains chromatin accessibility and promotes proliferative transcription during early regeneration, potentially modulating gene expression essential for cell plasticity and proliferation. This study identifies histone lactylation as a metabolic-epigenetic regulator of regenerative programs, providing mechanistic insights for the development of novel therapeutic strategies to enhance tissue repair.

T-cell lymphomas (TCLs) have been heterogeneous lymphoid malignancies with aggressive clinical phenotype and poor prognosis. Centrosomal protein 55 (CEP55) played a critical role in cytokinesis and served as a centrosome- and midbody-associated protein. Previous studies have reported the overexpression and clinical significance of CEP55 in various human malignancies, but the exact biological roles of CEP55 in TCLs remained unclear. In this study, we aimed to evaluate the CEP55 expression in patients with TCL and reactive hyperplasia of lymph nodes. The correlation between CEP55 levels and clinical characteristics was also explored for TCL patients. For further investigation, the cell viability of TCL cell lines after CEP55 inhibition was also assessed. Immunohistochemistry was applied to assess the elevated level of CEP55 in TCLs. After siRNA treatment, cell viability and apoptotic rate of TCL cell lines were observed with CCK-8 assay and flow cytometry, respectively. The Pearson's Chi-square test or Fisher's exact test was applied to analyze the correlations between CEP55 overexpression and clinical characteristics. All statistical tests were two-sided, and P < 0.05 was considered to be statistically significant. CEP55 was upregulated in TCL patients and significantly correlated with Ki-67 label index. Consistently, cell viability was decreased, and apoptosis was increased after the suppression of CEP55 in TCL cell lines. These results suggested that target CEP55 would be a novel therapeutic strategy for the TCL.

There is an urgent need for new therapeutic agents to treat patients with T-cell lymphoma (TCL). Multiple hematologic malignancies evade apoptosis through overexpression of anti-apoptotic proteins in the BCL-2 family, including BCL-2, BCL-XL, and MCL-1. We and others recently showed that a large fraction of cutaneous and peripheral TCL cell lines, patient-derived xenografts and primary patient samples depend on BCL-XL for survival (Koch et al, Blood. 2019; 133:566-575). These findings suggest that targeted inhibition of BCL-XL could offer therapeutic benefit for some TCL patients. Currently available small molecule BCL-XL inhibitors have failed during clinical development due to on-target and dose-limiting thrombocytopenia, as platelets depend on BCL-XL for survival. To overcome this toxicity, we developed DT2216, a novel proteolysis-targeting chimera (PROTAC) that targets BCL-XL to the Von Hippel Lindau (VHL) E3 ligase for proteasomal degradation. We selected the VHL ligase because platelets express very low levels of VHL, suggesting that they would be spared from the pro-apoptotic effects of DT2216. Here we examined the therapeutic potential of DT2216 against different TCL cell lines in vitro and in TCL xenograft mouse models. We first profiled the expression of different anti-apoptotic BCL-2 family proteins in multiple TCL cell lines (Fig. 1a) and tested their sensitivity to selective inhibitors for different BCL-2 family proteins as well as commonly-used chemotherapeutic agents (Table 1). The results showed that the TCL cells with higher levels of BCL-XL were more resistant to doxorubicin, etoposide and vincristine. DT2216 was more effective in reducing the viability of BCL-XL-dependent TCLs such as MyLa cells than ABT263 (a dual BCL-2/XL inhibitor). The EC50 value of DT2216 for MyLa TCL cells was less than 10 nM and DT2216 killed MyLa cells through induction of BCL-XL degradation and cellular apoptosis (Fig. 1b-f). Moreover, DT2216 was less toxic to human platelets than ABT263 in vitro with an EC50 &amp;gt; 3 μM. We further validated the effect and specificity of DT2216 in MJ cells, another BCL-XL-dependent TCL cell line, and demonstrated that its antitumor activity was dependent on proteasome activity. In vivo, when DT2216 was given to mice with MyLa TCL xenografts by i.p. injections at 10 mpk/q4d, it significantly inhibited tumor growth, whereas ABT263 at the same dose had no significant effect (Fig. 1g). More importantly, after MyLa xenografted mice failed to respond to ABT263 treatment, we subjected the mice to DT2216 (10 mpk/q4d), which induced rapid tumor regression and increased the survival of the mice without causing significant reduction of blood platelets (Fig. 1h). These effects were associated with a significant reduction in BCL-XL expression and activation of caspase cascade in tumor xenografts. However, some TCLs depend on not only BCL-XL but also BCL-2 or MCL-1 for evasion of apoptosis. Therefore, we next assessed the therapeutic potential of DT2216 in combination with a selective BCL-2 inhibitor using the TCL PDX DFTL-28776, which depends on both BCL-XL and BCL-2 for survival. We found that the combination of DT2216 with ABT199 (a selective BCL-2 inhibitor) could more effectively kill DFTL-28776 TCL cells than either agent alone in cell culture. The effect of the combination treatment on the growth of DFTL-28776 PDX in vivo is under examination. Collectively, our findings suggest that targeting BCL-XL using DT2216 can selectively kill BCL-XL-dependent TCL cells without causing significant platelet toxicity. Moreover, the combination of DT2216 with an inhibitor targeting other anti-apoptotic BCL-2 family proteins may have broad therapeutic utility against multiple TCL types and other cancers dependent on BCL-XL. Disclosures He: University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Khan:University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zhang:University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zheng:Dialectic Therapeutics: Equity Ownership, Other: Co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Weinstock:Celgene: Research Funding. Zhou:Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents.

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). Glycolysis plays a crucial role in PH pathogenesis, but the epigenetic mechanisms linking glycolysis to PASMCs proliferation remain unclear. Histone lactylation, a novel post-translational modification derived from glycolytic lactate, may regulate PASMCs proliferation. Primary rat PASMCs were cultured under hypoxia and treated with sodium L-lactate (NaLa) to assess glycolytic activity and histone lactylation. RNA sequencing, RT-qPCR, and Western blotting identified differentially expressed genes (DEGs), while ChIP-qPCR evaluated histone lactylation enrichment at gene promoters. In vivo, a hypoxia-induced PH rat model was used to examine the effect of glycolysis inhibition using oxamate. Mendelian randomization (MR) analysis assessed the causal relationship between placental growth factor (PGF) and PH. Hypoxia and NaLa treatment significantly increased glycolytic activity, lactate production, and histone lactylation, promoting PASMCs proliferation. Transcriptomic analysis identified 157 DEGs, with five key genes (Gbe1, Pgf, Mt2A, Ythdf2 and Gys1) upregulated in response to histone lactylation. ChIP-qPCR confirmed H3K18la enrichment at their promoters. Glycolysis inhibition with oxamate effectively reduced histone lactylation, PASMCs proliferation, and vascular remodeling in hypoxic PH rats. MR analysis identified PGF as a causal factor contributing to PH risk, suggesting a potential therapeutic target. This study reveals that glycolysis-induced histone lactylation drives PASMCs proliferation and vascular remodeling in PH. Targeting lactate metabolism and histone lactylation may provide a novel therapeutic approach.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11010-025-05342-8.

The Bruton’s Tyrosine Kinase Inhibitor CC-292 in Diffuse Large B-Cell Lymphoma (DLBCL), T-Cell Lymphoma (TCL), and Hodgkin Lymphoma (HL): Induction of Cell Death and Examination of Rational Novel/Novel Therapeutic Combinations

The Warburg effect, originally describing augmented lactogenesis in cancer, is associated with diverse cellular processes such as angiogenesis, hypoxia, macrophage polarization, and T-cell activation. This phenomenon is intimately linked with multiple diseases including neoplasia, sepsis, and autoimmune diseases1,2. Lactate, a compound generated during Warburg effect, is widely known as an energy source and metabolic byproduct. However, its non-metabolic functions in physiology and disease remain unknown. Here we report lactate-derived histone lysine lactylation as a new epigenetic modification and demonstrate that histone lactylation directly stimulates gene transcription from chromatin. In total, we identify 28 lactylation sites on core histones in human and mouse cells. Hypoxia and bacterial challenges induce production of lactate through glycolysis that in turn serves as precursor for stimulating histone lactylation. Using bacterially exposed M1 macrophages as a model system, we demonstrate that histone lactylation has different temporal dynamics from acetylation. In the late phase of M1 macrophage polarization, elevated histone lactylation induces homeostatic genes involved in wound healing including arginase 1. Collectively, our results suggest the presence of an endogenous “lactate clock” in bacterially challenged M1 macrophages that turns on gene expression to promote homeostasis. Histone lactylation thus represents a new avenue for understanding the functions of lactate and its role in diverse pathophysiological conditions, including infection and cancer.

Trka, a Novel Binding Partner of NPM-ALK Oncogenic Tyrosine Kinase, Facilitates the Survival of T-Cell Anaplastic Large-Cell Lymphoma