The switch of TCA cycle mode determines the fate of pirtobrutinib-tolerant persister cells

Abstract 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.