Regulation of ribosomal RNA synthesis in Acute Myeloid Leukemia Free

Hematopoiesis generates a complex hierarchy of cells with distinct functions, proliferation rates, and lifespans. A key determinant of hematopoietic cell identity is the precise control of protein and ribosome content. Ribosome biogenesis begins in the nucleoli with the production of specialized, non-coding ribosomal RNAs (rRNAs), which form the catalytic sites of ribosomes. Nascent 47S rRNA is transcribed from hundreds of tandem rRNA genes by RNA polymerase 1 (Pol1) and processed into mature 28S, 18S, and 5.8S rRNAs that are then used to assemble the 60S and 40S ribosomal subunits. rRNAs constitute more than 80% of total cellular RNA by mass, and rRNA synthesis accounts for 60% of the transcriptional activity in eukaryotic cells. Perturbing this process can lead to deleterious consequences on cell growth and differentiation, as observed in ribosomopathies such as Diamond-Schwachman syndrome and X-linked dyskeratosis congenita, underscoring its biological importance. However, the dynamics and regulation of rRNA synthesis in normal and diseased states of hematopoiesis remain poorly understood.

A pathognomonic feature of acute myeloid leukemia (AML) cells is their prominent nucleoli, and we hypothesized that AML cells are dependent on elevated rates of rRNA synthesis. Our group had previously published a “FISH-Flow” protocol for measuring cellular rRNA levels, in which fluorescent oligonucleotide probes are hybridized to nascent or mature rRNAs and quantified by flow cytometry (Antony et al. Molecular Cell 2022, STAR Protocols 2023). We applied this assay along with OP-puromycin labeling to map out relative rRNA abundances and protein translational capacities across the hematopoietic tree in bone marrow aspirates from healthy donors and newly diagnosed AML patients. Our results show that rRNA and protein synthesis rates vary widely throughout normal hematopoiesis, peaking in stem/progenitor cells (CMPs, GMPs, MEPs) and reaching a nadir in terminally differentiated cells. As predicted, AML cells possess significantly higher levels of nascent and mature rRNAs than any normal hematopoietic cell population (1.5- to 2-fold of stem/progenitor cells). Moreover, AML cells with the highest rRNA levels appear to exhibit less mature immunophenotypes. Analysis of publicly available single-cell RNA-seq data further confirmed that the Pol1 gene signature is overexpressed across genetically diverse AML subtypes.

To assess whether rRNA “hyper-synthesis” is essential for AML cell fitness and survival, we generated a HoxA9-ER mouse myeloid cell-line with POLR1A (the catalytic subunit of Pol1) fused to a FKBP12^F36V degron domain, which enables targeted degradation of Pol1 in the presence of a small molecule dTAG. Depletion of Pol1 led to a rapid decline in rRNA synthesis, followed by G1 cell cycle arrest and monocytic differentiation (as evidenced by increased Ly6C and decreased CD117 expression). Furthermore, dTAG treatment synergized with the Bcl-2 inhibitors venetoclax and navitoclax to induce apoptosis in the Pol1-degron cells. This synergy did not extend to standard AML chemotherapy agents (daunorubicin or cytarabine), indicating that there is a unique, synthetically lethal interaction between rRNA synthesis and the Bcl-2 signaling pathway in AML cells.

In summary, we show that rRNA synthesis is progressively downregulated during normal hematopoietic differentiation but aberrantly amplified in AML cells to support an oncogenic, stem-like state, allowing them to resist apoptotic stimuli and proliferate indefinitely. Notably, our findings highlight rRNA synthesis as a targetable vulnerability in AML, especially when combined with Bcl-2 inhibition.