Abstract Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved developmental program frequently co-opted in solid tumors to drive metastasis and therapy resistance. Whether analogous transcriptional and metabolic machinery operates in blood cancers has remained unexplored. Here, we demonstrate—for the first time—that drug-tolerant persister (DTP) cells in mantle cell lymphoma (MCL), a lethal non-Hodgkin’s lymphoma, exploit an EMT-like program distinct from cancer stem cell pathways to establish stable resistance to BTK inhibitors (BTKi) and anti-CD19 CAR T-cell therapy. Using pirtobrutinib (a clinically approved non-covalent BTKi), we established a reproducible, non-stochastic DTP cell model in MCL. Integrated RNA sequencing and ultra-high-resolution metabolomics revealed that therapeutic pressure triggers a striking morphogenetic switch: proliferative lymphoma cells transform into enlarged, quiescent “Giant cells” characterized by profound dedifferentiation and loss of B-cell identity (including CD19). Upon drug withdrawal, Giant cells rapidly revert to proliferative, normal-sized progeny, exposing a previously unrecognized reversible plasticity in hematologic malignancies. Mechanistically, DTP cells rewire the TCA cycle by engaging the malate-aspartate shuttle, sustaining anabolic metabolism during drug exposure. Drug removal abruptly shifts the TCA cycle to catabolic mode, fueling re-entry into cell cycles. This metabolic switch orchestrates global transcriptomic reprogramming and elevates acetyl-CoA levels, which stabilize the core EMT transcription factor SNAI1 via non-histone protein acetylation. Remarkably, acetylated SNAI1 translocates to nucleoli, driving explosive ribosome biogenesis (marked by fibrillarin upregulation)—a hallmark less studied in conventional EMT of epithelial cancers. Perturbing ATP-citrate lyase (ACLi), SNAI1, or fibrillarin disrupts this axis: ACLi/SNAI1 blockade accelerates exit from the Giant cell state, whereas fibrillarin loss traps cells in quiescence, collectively ablating DTP plasticity and restoring therapy sensitivity. In therapy-refractory MCL patients, DTP/Giant cells can be dynamically abundant—far exceeding classic minimal residual disease—and detectable by immunohistochemistry, metabolic imaging, and single-cell RNA sequencing. Thus, unlike solid tumors where EMT primarily enables invasion, blood cancers repurpose this ancient developmental program for metabolic resilience and immune evasion. Our work establishes that an EMT-like network, orchestrated by metabolic reprogramming and nucleolar SNAI1-driven ribosome biogenesis, governs DTP cell fate in MCL. Targeting this axis—particularly ribosome biogenesis—offers a transformative strategy to eradicate persister cells and overcome resistance to BTKi and CAR T-cell therapy. Citation Format: Wei Wang, Yang Liu, Heng-Huan Lee, Fangfang Yan, Yue Fei, Yijing Li, Chengtai Yu, Lin Tan, Lorenzi Phil, Qingsong Cai, Lei Nie, Michael Wang. EMT-like reprogramming drives drug-tolerant persister cell plasticity in mantle cell lymphoma via ribosome biogenesis [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 392.

Rett syndrome (RTT) is a neurological disorder caused by loss-of-function mutations in methyl-CpG-binding protein 2 (MECP2), which encodes a transcriptional regulator essential for maintenance of normal neuronal function. The current US Food and Drug Administration-approved treatment for RTT, trofinetide, mildly alleviates some symptoms. In contrast, reintroducing MeCP2 or increasing its amount through transgenesis in mouse RTT models improves most neurological phenotypes and enhances survival. Here, we devised a therapeutic strategy to moderately increase MeCP2 protein by modulating the alternative splicing of MECP2 to switch the less efficiently translated e2 to the more efficiently translated e1 isoform. We deleted Mecp2 exon 2 (unique to e2), leading to production of only e1 mRNA, and showed that this up-regulated MeCP2 by 50 to 60% in mice. Next, we investigated the consequences of isoform switching in two independent RTT induced pluripotent stem cell (iPSC)-derived neuron models harboring mutations that reduce both MeCP2 expression and function. Exon 2 deletion in neurons derived from patients with MeCP2-G118E up-regulated MeCP2, ameliorated morphological and electrophysiological changes, and corrected the dysregulated transcriptome in these neurons. Isoform switching in neurons derived from patients with MeCP2-G118E, modeling a severe RTT mutation, only modestly affected MeCP2 protein abundance and, despite this, led to a partial transcriptomic rescue. Last, an exon 2-skipping morpholino up-regulated MeCP2-E1 in vivo in mice. These data set the stage for a potential therapeutic strategy using antisense oligonucleotides to promote isoform switching in patients with RTT who carry partially functioning alleles of MECP2.

Abstract Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved developmental program frequently co-opted in solid tumors to drive metastasis and therapy resistance. Whether analogous transcriptional and metabolic machinery operates in blood cancers has remained unexplored. Here, we demonstrate—for the first time—that drug-tolerant persister (DTP) cells in mantle cell lymphoma (MCL), a lethal non-Hodgkin’s lymphoma, exploit an EMT-like program distinct from cancer stem cell pathways to establish stable resistance to BTK inhibitors (BTKi) and anti-CD19 CAR T-cell therapy. Using pirtobrutinib, a clinically approved non-covalent BTKi, we established a reproducible, non-stochastic DTP cell model in MCL. Integrated RNA sequencing and ultra-high-resolution metabolomics revealed that therapeutic pressure triggers a striking morphogenetic switch: proliferative lymphoma cells transform into enlarged, quiescent “Giant cells” characterized by profound dedifferentiation and loss of B-cell identity (including CD19). Upon drug withdrawal, Giant cells rapidly revert to proliferative, normal-sized progeny, exposing a previously unrecognized reversible plasticity in hematologic malignancies. Mechanistically, DTP cells rewire the TCA cycle by engaging the malate-aspartate shuttle, sustaining cytoplasmic anabolic metabolism during drug exposure. Drug removal abruptly shifts the TCA cycle to catabolic mode, fueling re-entry into the cell cycle. This metabolic switch orchestrates global transcriptomic reprogramming with elevated acetyl-CoA levels, which stabilizes the core EMT transcription factor SNAI1 via non-histone protein acetylation. Remarkably, acetylated SNAI1 translocated to nucleoli, driving ribosome biogenesis (marked by fibrillarin upregulation)—a hallmark less studied in conventional EMT of epithelial cancers. Perturbing ATP-citrate lyase (ACL), SNAI1, or inhibiting ribosome biogenesis disrupts this axis: ACL/SNAI1 blockade accelerates exit from the Giant cell state, whereas suppression of ribosome biogenesis leads to DTP cell death. In therapy-refractory MCL patients, DTP cells can be dynamically abundant, exceeding classic minimal residual disease—and detectable by immunohistochemistry, metabolic imaging, and single-cell RNA sequencing. Thus, unlike solid tumors where EMT primarily enables invasion, blood cancer cells repurpose this ancient developmental program for metabolic resilience and immune evasion. Our work establishes that an EMT-like network, orchestrated by metabolic reprogramming and nucleolar SNAI1-driven ribosome biogenesis, governs DTP cell fate in MCL. Targeting this axis—particularly ribosome biogenesis—offers a transformative strategy to eradicate persister cells and overcome resistance to BTKi and CAR T-cell therapy. Citation Format: Wei Wang, Yang Liu, Heng-Huan Lee, Fangfang Yan, Yijing Li, Yue Fei, Ching-Fei Li, Michael Wang. EMT-like reprogramming defines drug-tolerant persister cell plasticity in 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 A018.

Pirtobrutinib (PBN), a non-covalent BTK inhibitor, has been approved by the FDA for relapsed/refractory mantle cell lymphoma (MCL); however, resistance to PBN has been observed. To dissect the molecular dynamics driving PBN resistance, we performed integrative single-cell multi-omic profiling (scRNA-seq, scATAC-seq, and scDNA-seq) on longitudinal MCL patient samples. Our analyses revealed both genetic and non-genetic routes of resistance: In some patients, resistance involves sequential copy number gains (e.g., 1q, 2p, and 8q) accompanied by transcriptomic reprogramming and epigenetic alterations, whereas in others, resistance occurs through non-genetic mechanisms driven by transcriptional and epigenetic remodeling. Integration of scATAC-seq and scRNA-seq enabled gene regulatory network inference and in silico perturbation analysis, highlighting RAD21 and SMC3, core components of the cohesin complex, as chromatin regulators whose downregulation may re-sensitize cells to PBN. A stem-like malignant B cell population enriched in resistant samples exhibited features of metabolic reprogramming and epithelial-mesenchymal transition. Together, our findings highlight heterogeneous, multi-layered mechanisms of PBN resistance and suggest chromatin regulators as potential therapeutic targets to overcome PBN resistance in MCL.

Abstract The receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is highly expressed in hematologic and solid tumors and further upregulated during relapses following targeted or immunotherapies. In non-Hodgkin B-cell malignancies, ROR1 upregulation during disease progression contributes to relapses after CD19-CAR T-cell therapy, highlighting ROR1 as a compelling therapeutic target in mantle cell lymphoma (MCL). In this work, we demonstrate that ROR1 is markedly elevated in MCL and ROR1+ tumor cells become progressively enriched as the disease advances, particularly in specimens resistant to CD19-CAR T therapy. These observations reveal a clear association between heightened ROR1 level and relapse following CD19-CAR T-cell therapy, identifying ROR1 as an actionable vulnerability for therapeutic intervention in refractory/relapsed MCL, positioning ROR1-targeted bispecifics as a promising and clinically actionable approach to overcoming CAR T-cell resistance. To leverage this opportunity, we developed ROR1-directed bispecific modalities (ROR1-bsAb) for patients with CAR T-refractory disease and confirmed their functional activities. Engagement of healthy-donor T cells with the ROR1-bsAb markedly enhanced their effector function and induced robust, dose-dependent tumoricidal activity against ROR1high MCL cells, including primary MCL cells from CD19-CAR T-resistant patients, highlighting its therapeutic potential in overcoming CAR T-cell therapy relapses. Critically, while CD19-CAR T cells alone exhibited limited target specific tumoricidal activity, engagement with the ROR1-bsAb enabled CD19-CAR T cells to mediate robust and dose-dependent killing of ROR1high CAR T-resistant specimens. Given the emergence of CD19-independent CAR T cell-resistance, incorporating an alternative antigen-targeted strategy may overcome immune escape. Indeed, adding a Fc-less ROR1-bispecific significantly enhanced the antitumor activity of CD19-CAR T cells against both CD19-diminished Z-138-R and CAR T-resistant primary MCL. Consistently, Transwell antibody delivery from ROR1-bispecific-secreting T cells markedly increased cytokine production and reinstated the cytotoxicity against CAR T therapy-resistant MCL. We next validated the therapeutic potential of ROR1-bsAb in a cell line-derived xenograft model of MCL. Consistently, co-administration of either one of the ROR1-bispecific modalities and T cells elicited robust anti-lymphoma efficacy, as evidenced by pronounced suppression of tumor growth and prolonged survival. These findings highlight ROR1-targeted bispecific therapeutics as a clinically translatable strategy with strong potential to address the clinical unmet need. Taken together, this ROR1-directed bispecific platform, whether used either as a standalone modality or in combination with CAR T-cell therapy, offers considerable promise for overcoming ROR1-upregulation and antigen-loss-mediated resistance, ultimately improve outcomes for patients who relapse after CD19-CAR T-cell therapy. Citation Format: Yixin Yao, Fangfang Yan, Xiaolin Li, Yijing Li, Yue Fei, Joseph McIntosh, Wei Wang, Heng-Huan Lee, Yang Liu, Michael Wang. ROR1 upregulation after CD19-CAR T-cell relapse in mantle cell lymphoma reveals a targetable vulnerability for ROR1-directed bispecific modalities [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 A004.

Background Drug-tolerant persister (DTP) cells are increasingly recognized as a key contributor to therapy resistance in cancer patients. In mantle cell lymphoma (MCL), the mechanisms by which DTP cells adapt to treatments and develop resistance remain poorly understood. This study investigates the presence and development of pirtobrutinib-tolerant persister cells in MCL and aims to elucidate their underlying mechanisms of resistance. By identifying these mechanisms, we seek to provide insights into novel therapeutic strategies for targeting DTP cells in MCL patients. Methods A non-stochastic drug-tolerant persister (DTP) cell line model was developed using mantle cell lymphoma (MCL) cells with acquired resistance to therapies. This model includes Mino cells resistant to venetoclax (Mino-VEN-R) and JeKo-1 cells resistant to ibrutinib (JeKo-1-IBN-R). Both bulk and single-cell RNA sequencing were employed to characterize upregulated and downregulated pathways. Metabolite profiling was conducted using ultra-high-resolution mass spectrometry (HRMS). Oxygen consumption was measured with the Seahorse XF96 analyzer (Agilent Technologies). Anti-CD19 CAR T-cells were generated from human primary pan-T cells isolated from healthy donors, transduced with lentivirus expressing a CD19-directed CAR (FMC63 scFv, 4-1BB costimulatory domain, and CD3ζ stimulatory domain). The presence of pirtobrutinib-tolerant persister cells in MCL was validated using patient-derived organoid and xenograft models. Transcriptome profiles from 62 primary MCL patient samples were analyzed via RNA sequencing. Results MCL cells with abnormal morphology have been recognized as an indicator of aggressive diseases with poor response to therapies. However, less was known about their origin and mechanism. Here, we observed that pirtobrutinib treatment induces anabolic growth rather than proliferation in Mino-VEN-R and JeKo-1-IBN-R mantle cell lymphoma (MCL) cells, resulting in enlarged cells with pleomorphism, designated as “Giant cells.” Upon pirtobrutinib withdrawal, these cells resume proliferation, indicating a reversible phenotypic transition characteristic of a drug-tolerant persister (DTP) state, with Giant cells serving as a transitional state for pirtobrutinib-tolerant persister cells. RNA sequencing revealed that Giant cells exhibit a dedifferentiated state under pirtobrutinib treatment, marked by significant loss of B-cell markers, such as CD19, conferring resistance to anti-CD19 CAR T-cell therapy in NSG mice. Mechanistically, an anabolic tricarboxylic acid (TCA) cycle fuels DTP cell development, driven by elevated acetyl-CoA levels mediated by increased ATP-citrate lyase (ACL) activity, which profoundly alters gene expression profiles. GOT2, a key enzyme, supports biosynthesis, including nucleotide production, while ribosome biogenesis is a hallmark of the Giant cell state. Notably, the EMT modulator SNAI1 is a key transcription factor potentially driving dedifferentiation. Upon drug withdrawal, the TCA cycle shifts to a catabolic mode, as validated in patient-derived organoid (PDO) cultures. Additionally, DTP cells in the Giant cell state were identified in patient-derived xenograft (PDX) tumor samples from an MCL patient resistant to pirtobrutinib and CAR T-cell therapy. More evidence from primary MCL patients with resistance to pirtobrutinib and CAR T-cell therapies showed the potential presence of DTP cells, including results from IHC imaging and RNA sequencing. These indicate that DTP cells can be more prevalent in therapy-resistant MCL patients compared to therapy-sensitive patients. Conclusions We conclude that non-genetic mechanisms, particularly metabolic reprogramming, drive the fate of drug-tolerant persister (DTP) cells in mantle cell lymphoma (MCL). The tricarboxylic acid (TCA) cycle dynamically switches between anabolic and catabolic modes, enabling tumor cells to adapt and evade therapeutic stress. Acetyl-CoA is a critical metabolite that reshapes the epigenetic landscape of tumor cells. This phenotypic plasticity of DTP cells contributes to tumor heterogeneity following prolonged therapies. DTP cells are prevalent in MCL patients with therapy resistance. Targeting the dedifferentiated state of DTP cells, rather than oncogenic lesions, offers a novel strategy to overcome stable therapy resistance in MCL.

ABSTRACT:Bruton tyrosine kinase inhibitors (BTKis) and cell therapy have successfully been used to treat mantle cell lymphoma (MCL). However, therapy resistance inevitably emerges. Cancer cells can progressively develop stable resistance by traversing through a transient drug-tolerant persister (DTP) state. The mechanisms enabling DTP cells to reversibly adapt to therapies and evolve to acquire heterogeneity remain poorly understood, and characterizing DTP cells in MCL continues to pose a challenge for clinic translation. Here, using pirtobrutinib, a recently US Food and Drug Administration-approved noncovalent BTKi, we identified pirtobrutinib-tolerant persister cells exhibiting morphological variability by presenting a unique population of enlarged cells (giant cells) with reversible fate transitions. During treatment, giant cells enter a nonproliferative, dedifferentiated state, addicted to an activated cytosolic tricarboxylic acid (TCA) cycle coupled with the malate-aspartate shuttle to engage in biosynthesis. Upon drug removal, the TCA cycle shifts to oxidative catabolism, promoting giant cells to differentiate into regular-sized cells. Throughout the transition, acetyl coenzyme A modulates cell fate by fine-tuning stemness. Our biphasic model demonstrates that the metabolic switch governs the phenotypic plasticity of DTP cells in MCL, resulting in a dynamic presence of DTP cells across various developmental states in response to systemic therapies. Targeting giant cells before their differentiation offers a promising strategy to overcoming therapy resistance in MCL.

Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene cause Rett syndrome, a severe childhood neurological disorder. MeCP2 is a well-established transcriptional repressor, yet upon its loss, hundreds of genes are dysregulated in both directions. To understand what drives such dysregulation, we deleted Mecp2 in adult mice, circumventing developmental contributions and secondary pathogenesis. We performed time series transcriptional, chromatin, and phenotypic analyses of the hippocampus to determine the immediate consequences of MeCP2 loss and the cascade of pathogenesis. We find that loss of MeCP2 causes immediate and bidirectional progressive dysregulation of the transcriptome. To understand what drives gene downregulation, we profiled genome-wide histone modifications and found that a decrease in histone H3 acetylation (ac) at downregulated genes is among the earliest molecular changes occurring well before any measurable deficiencies in electrophysiology and neurological function. These data reveal a molecular cascade that drives disease independent of any developmental contributions or secondary pathogenesis.

Mitochondrial miRNAs (mitomiRs), which are miRNAs that located within mitochondria, have emerged as crucial regulators in a variety of human diseases, including multiple types of cancers. However, the specific role of mitomiRs in clear cell renal cell carcinoma (ccRCC) remains elusive. In this study, we employed a combination of experimental and bioinformatic approaches to uncover the diverse and abundant subcellular distribution of miRNAs within mitochondria in ccRCC. Notably, RNA sequencing after mitochondrial fractionation identified miR-134-5p as a miRNA predominantly detected in the mitochondria of 786O cells, and its expression is significantly upregulated compared to that in 293T cells. Differential expression and survival analyses from TCGA reveal that the upregulation of miR-134-5p is prevalent and closely associated with poor survival outcomes in ccRCC patients. Functionally, exogenous overexpression of miR-134-5p mimics promotes migration in both 786O and Caki-1 cells. Mechanistically, overexpressing the miR-134-5p mimic dramatically downregulates the mRNA levels of CHST6, SFXN2, and GRIK3, whereas the miR-134-5p inhibitor markedly upregulates their expression. Notably, these target mRNAs also predominantly detected in the mitochondria of 786O cells. The downregulated expression signatures of CHST6, SFXN2, and GRIK3 are also closely correlated with poor survival outcomes in ccRCC patients. Taken together, our work identifies a novel mitomiR, miR-134-5p, in ccRCC, provides potential targets that could serve as effective biomarkers for ccRCC diagnosis and prognosis, and opens new avenues for understanding the mitomiR-directed regulatory network in ccRCC progression.

BACKGROUND:Tumour immunotherapy holds great promise as a treatment for cancer, which ranks as the second highest cause of mortality worldwide. This therapeutic approach can be broadly categorized into two main types: active immunotherapy and passive or adoptive immunotherapy. Active immunotherapy, such as cancer vaccines, stimulates the patients' immune system to target tumour cells. On the other hand, adoptive immunotherapy involves supplying in vitro activated immune cells, such as T cells, natural killer cells and macrophages, to the patient to combat the tumour. Induced pluripotent stem cells are extensively utilized in both active and adoptive tumour immunotherapy due to their pluripotency and ease of gene editing. They can be differentiated into various types of immune cells for direct cancer treatment and can also function as tumour vaccines to elicit an immune response against the tumour. Importantly, iPSCs can be leveraged to develop off-the-shelf allogenic immunotherapy products. CONCLUSION:This article provides a comprehensive review of the application of iPSCs in tumor immunotherapy, along with a discussion of the opportunities and challenges in this evolving field.

Lung cancer is the leading cause of cancer-related death and has the second-highest incidence worldwide. For patients with advanced EGFR-mutated non-small cell lung cancer, EGFR tyrosine kinase inhibitors (EGFR-TKIs) are the preferred treatment option; however, acquired resistance to TKIs is inevitable. Gefitinib and osimertinib, the first-generation and third-generation EGFR-TKI, have shown promising results in patients with EGFR-mutated lung cancer in clinical treatment. Here, we identified that pyruvate dehydrogenase kinase 1 (PDK1) was up-regulated in gefitinib- and osimertinib-resistant cell lines, and PDK1 knockdown rendered cells more sensitive to TKI treatment. PDK1 expression levels were significantly increased in lung, colon, liver, and breast cancer tissues compared with those in normal tissues. Histone demethylase KDM3A was also induced in TKI-resistant cell lines, and demethylated histone H3 lysine 9 to facilitate PDK1 expression to regulate TKI resistance. Further study demonstrated that METTL16 promoted the m6A modification of PDK1 mRNA, and the m6A reader IGF2BP1 directly recognized and enhanced PDK1 mRNA stability. Interestingly, KDM3A also induced METTL16 expression. Moreover, PDK1 inhibitor JX06 rendered cancer cells more sensitive to gefitinib treatment in vivo, and JX06 and gefitinib combination treatments have a synergic effect to inhibit tumor growth. In conclusion, the KDM3A/METTL16/PDK1 axis plays an important role in cancer development and TKI resistance, which may offer new prognostic biomarkers and therapeutic targets for TKI resistance in the future.

Abstract Successive generations of BTK inhibitors and CAR T-cell therapy have transformed the treatment of mantle cell lymphoma (MCL). However, treatment resistance has inevitably emerged, creating significant medical need. Recently a novel, clinical stage small molecule, BTM-3566, has been described that targets a mitochondrial process essential for Diffuse Large B-cell lymphoma (DLBCL) survival. BTM-3566 activates the mitochondrial protease OMA1, triggering the ATF4 integrated stress response (ISR). BTM-3566 has robust in vitro efficacy in DLBCL cell lines irrespective of genomic background and elicits complete tumor regression in multiple CDX and PDX mouse models of DLBCL. Based on the activity observed in DLBCL, we further explored the effectiveness of BTM-3566 in MCL models in vitro and in xenograft mouse models in vivo. BTM-3566 inhibited cell proliferation across a panel of MCL cell lines, irrespective of resistance status to ibrutinib and venetoclax (IC50s of 142.5-687.6 nM). Following a 24-hour treatment with BTM-3566, a dose-dependent increase in apoptosis was observed in four tested MCL cell lines—JeKo-1, JeKo-IBN-R, Mino, and Mino-VEN-R, as determined by increased caspase-3 activation and PARP cleavage. Similarly, western blot analysis revealed dose-dependent reductions in MCL-1 and c-MYC, accompanied by an increase in the transcription factor ATF4, indicative of activation of the stress response. We evaluated the in vivo efficacy of BTM-3566 in the JeKo-CDX mouse model. Daily oral administration of BTM-3566 at 10, 20 and 30 mg/kg (n = 5 for each group) in a 5-day on and 2-day off schedule resulted in tumor regression at all three doses (Treatment vs. Vehicle, p < 0.01). We expanded our investigation to PDX mouse models, including those derived from patients resistant to multiple therapies. Four PDX models encompassing naive, rituximab-resistant, BTKi-CAR T dual-resistant and BTKi-VEN-CAR T triple-resistant models were evaluated. BTM-3566 treatment results in nearly complete tumor growth inhibition associated with robust reductions in tumor burden in treated mice across all four PDX models (reduction in tumor weight at the study endpoint, p < 0.001). Notably, no toxicity was observed during the treatment period. In summary, BTM-3566 is effective in treating MCL in vitro and in vivo in preclinical studies. Citation Format: Yang Liu, Heng-Huan Lee, Joseph McIntosh, Yijing Li, Tianci Zhang, Wei Wang, Matthew J. Kostura, Michael Wang. The integrated stress activator BTM-3566 overcomes therapeutic resistance in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 676.

Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by frequent relapses. The development of resistance to ibrutinib therapy remains a major challenge in MCL. We previously showed that glutaminolysis is associated with resistance to ibrutinib. In this study, we confirmed that glutaminase (GLS), the first enzyme in glutaminolysis, is overexpressed in ibrutinib-resistant MCL cells, and that its expression correlates well with elevated glutamine dependency and glutaminolysis. Furthermore, we discovered that GLS expression correlates with MYC expression and the functioning of the glutamine transporter ASCT2. Depletion of glutamine or GLS significantly reduced cell growth, while GLS overexpression enhanced glutamine dependency and ibrutinib resistance. Consistent with this, GLS inhibition by its specific inhibitor telaglenastat suppressed MCL cell growth both in vitro and in vivo. Moreover, telaglenastat showed anti-MCL synergy when combined with ibrutinib or venetoclax in vitro, which was confirmed using an MCL patient-derived xenograft model. Our study provides the first evidence that targeting GLS with telaglenastat, alone or in combination with ibrutinib or venetoclax, is a promising strategy to overcome ibrutinib resistance in MCL.

Background Covalent Bruton's tyrosine kinase (BTK) inhibitors have revolutionized the therapy of mantle cell lymphoma (MCL). In January 2023, the reversible BTK inhibitor pirtobrutinib was approved by the FDA for the treatment of relapsed or refractory MCL. Nemtabrutinib is another reversible BTK inhibitor which also targets other kinases. The objective of this study was to evaluate the efficacy of nemtabrutinib alone and in combination with anti-CD19 CAR T cells against MCL. Methods Cell viability was measured following a 72-hour treatment with nemtabrutinib in a panel of MCL cell lines using CellTiter-Glo luminescent cell viability assay (Promega). Annexin V/PI staining was utilized to determine whether nemtabrutinib induces cell death through apoptosis. Patient-derived organoids (PDOs) were generated by culturing patient primary cells in 50% Matrigel (Corning) and cultured in cytokine-containing medium. The PDOs were treated for 72 hours, and viability assays were performed. Western blotting was employed to investigate the drug impact on kinase signaling pathways. Nemtabrutinib treated Mino cells were subjected to bulk RNA sequencing to interrogate the transcriptome profiling. The anti-MCL efficacy of nemtabrutinib was tested in an ibrutinib-resistant patient derived xenograft (PDX) mouse model in vivo. Additionally, the potential synergistic effects of nemtabrutinib and anti-CD19 CAR T cells were examined using the luciferase-expressing MCL cells. Results Nemtabrutinib demonstrated comparable growth inhibitory activity to ibrutinib in MCL cell lines with IC 50 values at micromolar concentrations (IC 50 = 0.7-10.1 μM). Remarkably, in the 3D PDO screening system that mimics the tumor microenvironment for MCL, nemtabrutinib displayed superior anti-MCL efficacy compared to ibrutinib (p < 0.01). The apoptotic potential of nemtabrutinib was further investigated using Annexin V/PI staining, revealing a dose-dependent induction of apoptosis, and demonstrated a similar sensitivity profile across cell lines observed with ibrutinib. Western blotting revealed that nemtabrutinib effectively inhibited phosphorylation at both Tyr223 and Tyr551 sites of BTK, and also dramatically suppressed the activation of Src family kinases, Syk and ERK. Bulk RNA-seq was performed to examine the transcriptome profile following nemtabrutinib treatment. 449 genes were identified to be significantly upregulated and 460 genes were downregulated. Gene set enrichment analysis uncovered a significant decrease in TNFα signaling via NF-κB, inflammatory response, and IFNγ response signalings (FDR < 0.05). In an ibrutinib-resistant PDX mouse model, treatment with nemtabrutinib resulted in a significant reduction in tumor burden and effectively attenuated the tumor involvement in spleen, liver and bone marrow. Furthermore, to enhance efficacy, we investigated the combination of nemtabrutinib with anti-CD19 CAR T cells in luciferase-expressing MCL cells. The result demonstrated that nemtabrutinib enhanced the effector function and anti-MCL activity of anti-CD19 CAR T cells. Conclusion Nemtabrutinib demonstrated favorable anti-MCL efficacy in both in vitro and in vivo studies, the promising synergistic effects observed in combination with CAR T cells warrants further investigation in both preclinical and clinical settings.

Bruton’s tyrosine kinase (BTK) is a proven target in mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma. However, resistance to BTK inhibitors is a major clinical challenge. We here report that MALT1 is one of the top overexpressed genes in ibrutinib-resistant MCL cells, while expression of CARD11, which is upstream of MALT1, is decreased. MALT1 genetic knockout or inhibition produced dramatic defects in MCL cell growth regardless of ibrutinib sensitivity. Conversely, CARD11-knockout cells showed antitumor effects only in ibrutinib-sensitive cells, suggesting that MALT1 overexpression could drive ibrutinib resistance via bypassing BTK/CARD11 signaling. Additionally, BTK knockdown and MALT1 knockout markedly impaired MCL tumor migration and dissemination, and MALT1 pharmacological inhibition decreased MCL cell viability, adhesion, and migration by suppressing NF-κB, PI3K/AKT/mTOR, and integrin signaling. Importantly, cotargeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in ibrutinib-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that MALT1 overexpression associates with resistance to BTK inhibitors in MCL, targeting abnormal MALT1 activity could be a promising therapeutic strategy to overcome BTK inhibitor resistance, and cotargeting of MALT1 and BTK should improve MCL treatment efficacy and durability as well as patient outcomes.