Efficacy and mechanism of BTK degrader nx-5948 in secondary hemophagocytic lymphohistiocytosis

Abstract Background: Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory condition marked by fever, cytopenia, hepatosplenomegaly, and multi-organ failure. Bruton's tyrosine kinase (BTK) is a key driver of NF-kB activation and an important target in HLH. BTK inhibitor zanubrutinib has demonstrated certain therapeutic benefits in HLH. However, the kinase inhibition may not completely block NF-κB activation, and the residual cytokine storm can undermine their overall effectiveness. NX-5948 is a novel oral small molecule that induces BTK degradation by recruiting the cereblon E3 ligase complex and has shown potential in other diseases. Here, we report that the BTK degrader NX-5948 can treat HLH by modulating macrophage polarization and mitigating cytokine storm. Material and method: In vitro, murine macrophage cell line RAW264.7 was stimulated with CpG plus IFN-γ to establish an HLH-like inflammatory model. Subsequently,the anti-inflammatory potency of the JAK inhibitor ruxolitinib, BTK inhibitors ibrutinib and zanubrutinib, as well as BTK degrader NX-5948 was compared. CCK-8, annexin V/PI flow cytometry and cell cycle flow cytometry were employed to quantify the selective induction of macrophage apoptosis by the individual drugs. Flow cytometry and ELISA were then applied to assess drug-induced changes in macrophage polarization, ROS production, and cytokine release. Western blot analysis was used to assess the activation of the BTK/NF-κB pathway. In vivo, a secondary HLH model was induced in C57/BL6 mice by repeated injections of CpG. The therapeutic efficacy and systemic toxicity of the individual drugs were evaluated by assessing complete blood counts, hepatic function markers, renal function markers, the ratios of liver and spleen weight to body weight, serum cytokine levels, and hematoxylin and eosin (H&E) staining. To explore and validate the pharmacological mechanisms of NX-5948 in HLH, RNA sequencing, immunohistochemistry, and western blot analyses were employed. Results: In vitro, CCK-8 assays demonstrated that NX-5948 significantly inhibited macrophage proliferation, exhibiting a lower IC50（0.14μM） than ruxolitinib, ibrutinib, and zanubrutinib. Apoptosis and cell cycle analyses revealed that NX-5948 potently induced macrophage apoptosis and arrested cells at S/G2 phase. Furthermore, NX-5948 reduced ROS production and cytokine release (TNF-α, IL-1β, IL-6), as well as prevented M1 polarization and promoted M2 polarization. The effects of NX-5948 superior to comparator drugs at equivalent concentrations. Western blot analysis confirmed BTK degradation by NX-5948 and suppressed phosphorylation of key BTK/NF-κB pathway proteins (BTK, PLCγ2, IκBα, NF-κB). In vivo, NX-5948 significantly ameliorated hepatosplenomegaly and elevated platelet counts in secondary HLH mouse models. It concurrently attenuated hepatic and renal dysfunction. NX-5948 also reduced serum levels of TNF-α, IL-6, IL-1β, ferritin, and soluble interleukin-2 receptor (sIL-2R). Histopathological examination by H&E staining revealed no pathological alterations in major organs following NX-5948 treatment. RNA sequencing demonstrated significant upregulation of the BTK/NF-κB pathway in model mice versus controls, which was attenuated by NX-5948. Immunohistochemical and Western blot analyses confirmed potent inhibition of p-BTK, p-PLCγ2, p-IκBα, and p-NF-κB activation. Conclusion: The BTK degrader NX-5948 exerted potent therapeutic effects against HLH pathogenesis in both in vitro and in vivo models. These findings indicate that BTK degraders represent a novel therapeutic strategy for the clinical management of HLH.