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IntroductionBromodomain and Extraterminal (BET) proteins play a crucial role in cellular proliferation and differentiation through the epigenetic regulation of gene transcription. As a result, inhibiting BET family proteins emerges as a promising epigenetic approach for treating various cancers. However, clinical trials have indicated that thrombocytopenia is a dose-limiting toxicity associated with BET inhibition. This study aims to explore the mechanism and clinical pharmacology of BMS-986158-induced thrombocytopenia and to identify biomarkers as tools to identify patients at higher risk, thereby better managing toxicity and improving efficacy.MethodsBlood samples from preclinical rats and clinical trial patients treated with BMS-986158 were collected for transcriptional expression profiling. Target engagement was confirmed by measuring HEXIM1 and monitoring thrombocytopenia following BET inhibition. Genes regulated by GATA1 and associated with thrombopoiesis, including NFE2 and PF4, were investigated. The outcomes of the rat and human studies were compared to identify biomarkers for the early prediction of thrombocytopenia associated with BET inhibition.ResultsTarget engagement was confirmed with dose-dependent responses of HEXIM1 expression and platelet counts. Blood samples from rats treated with BMS-986158 showed dose-dependent downregulation of GATA1, NFE2, and PF4 at 24 h or earlier post-treatment. Similarly, patients’ blood samples collected within 24 h post-treatment with BMS-986158 also showed dose-dependent downregulation of GATA1 and PF4 in all treated groups. Significant downregulation of PF4 and NFE2 genes was found in patients with low platelet counts. A strong correlation between the expression of GATA1 and the genes NFE2 and PF4 was observed in both preclinical and clinical studies.DiscussionThe consistent downregulation of GATA1, NFE2, and PF4 transcription within hours post-BMS-986158 treatment in both preclinical and clinical studies demonstrates that BET inhibitors induce thrombocytopenia by altering GATA1 gene expression and its downstream genes, NFE2 and PF4, which regulate megakaryopoiesis and thrombopoiesis. Early detection of transcriptional changes in blood samples during treatment courses positions NFE2 and PF4 as promising biomarkers for proactively monitoring and mitigating treatment-emergent thrombocytopenia. Graphical abstract Flowchart illustrating the effects of BET inhibitors. BET inhibitor impacts BRD2/BRD4, linked to GATA1 hematopoietic transcription factor. GATA1 affects NFE2 and PF4, influencing thrombopoiesis and leading to thrombocytopenia. Separately, BRD2/BRD4 affects oncogenes, including MYC, related to cancer.

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult.

Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F-dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F-binding regulate JAK2-mediated signals in MPNs.