Decoding Thrombopoiesis: Insights from RNA Polymerase I Inhibition

Abstract

Platelets are the primary mediators of haemostasis, maintaining vascular integrity. Thrombocytopenia (a low platelet number in blood) is a common clinical observation. Current therapies for thrombocytopenia have limitations, highlighting a need for novel therapeutics. RNA polymerase I (Pol I) transcription is essential for ribosome biogenesis (Ribi), a process crucial for protein production. Pol I transcription positively regulates cellular proliferation pathways and is often upregulated in cancer. Inhibiting Pol I triggers p53-dependent and independent apoptosis. CX-5461, a first-in-class Pol I inhibitor, has demonstrated efficacy in Phase I/II trials. During preclinical testing, CX-5461 was shown to increase circulating mouse platelet numbers. This thesis aims to elucidate the mechanism(s) by which CX-5461 and other Ribi inhibitors elevate platelet numbers using experimental models in vitro and in vivo. In a first-in-human dose-escalation trial, a single dose of CX-5461 increased platelet counts (up to 34%) in 9/16 haematological malignancy patients. C57BL/6 mice were orally administered Ribi inhibitors, and blood cell numbers were measured, along with platelet receptors and activation markers and platelet production indices. Treatment with CX-5461, PMR-116 or BMH-21 led to a rapid, >1.5-fold increase in platelet counts by day 7, with sustained supraphysiological platelet levels reversible by treatment cessation. Flow cytometry and functional assays revealed normal platelet receptor levels and function. In a panel of 23 cytokines, serum from CX-5461-treated mice had only reduced C-C motif chemokine ligand (CCL) 5 and increased CCL11. Corresponding BM supernatants showed increased CCL11, interleukin (IL) 1-a, monocyte chemoattractant protein 1, CCL5, and keratinocyte chemoattractant. Haematopoietic cell subpopulations were immunophenotyped from murine bone marrow and cell cultures ex vivo. Inhibitor treatment showed normal megakaryocyte (MK) ploidy status and proplatelet formation. However, CX-5461 treatment induced a >2-fold increase in BM and splenic MK numbers and increased Sca1+MK, suggesting a non-canonical haematopoietic pathway activation. Non-canonical MK-biased haematopoiesis is characterised by directly differentiating haematopoietic stem/progenitor cells into MKs, bypassing oligopotent progenitors. MK colony-forming unit analysis revealed a significant increase in the total number of MK colonies. CX-5461 treatment increased BM MK-biased multipotent progenitor-2 cells (LSKCD135-CD48+CD150+). Plasma TPO (ELISA) and liver TPO mRNA levels (RT-qPCR) were unchanged. TPO receptor-deficient mice and carboplatin-induced thrombocytopenia models were employed to assess the therapeutic potential of CX-5461 in managing thrombocytopenia. Administration of CX-5461 in both models significantly increased platelet counts, indicating that CX-5461 promotes platelet production independently of the TPO receptor and accelerates platelet recovery following a nadir. To elucidate the mechanism of CX-5461-induced platelet production, single-cell RNA sequencing (scRNAseq) of 15,000 fluorescence-activated cell sorted LSK cells (50,000 reads/cell) from mice treated with either vehicle or CX-5461 for 2 weeks was performed. Gene expression analysis revealed that CX-5461 treatment increases p53 signalling and P-selectin gene expression features similar to MK-biased HSCs and myeloid-biased haematopoiesis. RNA velocity analysis showed a marked increase in the association of MK progenitor cells with mature MKs following treatment. CX-5461 treatment appears to increase circulating platelet levels by stimulating the MK-biased haematopoietic pathway independently of previously known triggers such as TPO. Ribi inhibitors offer a novel foundation for developing thrombopoietic drugs and provide a tool to further our understanding of both steady-state and stress thrombopoiesis. Show more