The ribosome biogenesis inhibitor CX-5461 increases platelet count in humans and enhances murine megakaryopoiesis

Abstract Treatment of sub-optimal platelet production and thrombocytopenia remains a major clinical challenge and thrombocytopenia is a common and serious complication of anticancer therapies. Here, we identify a novel thrombopoietic activity of the first-in-class ribosome biogenesis inhibitor CX-5461. In a phase I trial, 56% (9/16) of patients with haematological malignancy (myeloma, lymphoma, or leukaemia) exhibited up to a 34% increase in platelet count following a single dose of CX-5461. C57BL/6 or thrombopoietin (TPO)-receptor-deficient mice received three doses/week of 35 mg/kg CX-5461 or vehicle. CX-5461 elicited a rapid, reversible, and sustained ~1.7-fold increase in platelet numbers at day 7 without altering platelet function, lifespan, or levels of inflammatory cytokines. New platelets were increased 2.3-fold. Bone marrow analysis revealed a specific 2-fold expansion of megakaryocytes (MKs), increased Sca1⁺ MKs (p<0.01), and enrichment of MK-biased multipotent progenitor-2 (p=0.015) and MPP3 (p<0.0001), independent of TPO or c-mpl signalling. MK ploidy levels remained comparable. CX-5461 treatment induced a >3-fold increase in MK-CFU. CX-5461 induced thrombopoiesis without altering plasma or liver TPO mRNA levels and increased platelet counts 5-fold (p<0.0005) in TPO receptor-/- mice. CX-5461 treatment reduced the depth of carboplatin-induced thrombocytopenia and accelerated platelet recovery. Single-cell RNA sequencing and RNA velocity analysis confirmed enhanced differentiation of MK progenitors. These findings demonstrate that inhibition of ribosome biogenesis promotes TPO-independent megakaryopoiesis and identifies a previously unrecognised therapeutic opportunity to support platelet recovery in cancer treatment and potentially other thrombocytopenic states. CX-5461 not only exerts potent anti-tumour activity but also significantly enhances megakaryopoiesis and increases platelet counts in both humans and mice, through a mechanism that is independent of TPO or c-mpl receptor signalling. Key findings were replicated in mice treated with other ribosomal biogenesis inhibitors such as BMH-21. RNA-Polymerase I inhibition offer a novel target for treating thrombocytopenia and a tool to further our understanding of thrombopoiesis. This unique property distinguishes CX-5461, and other inhibitors of RNA Pol I transcription from other anticancer agents and highlights this class of reagent to have potential dual utility as both antineoplastic and thrombopoietic agents, particularly in settings of chemotherapy-induced thrombocytopenia or in patients with impaired TPO signalling.