HEATR3 variants impair nuclear import of uL18 (RPL5) and drive Diamond-Blackfan anemia.

The congenital bone marrow failure syndrome Diamond-Blackfan anemia (DBA) is typically associated with variants in ribosomal protein (RP) genes impairing erythroid cell development. Here we report multiple individuals with biallelic HEATR3 variants exhibiting bone marrow failure, short stature, facial and acromelic dysmorphic features, and intellectual disability. These variants destabilize a protein whose yeast homolog is known to synchronize the nuclear import of RPs uL5 (RPL11) and uL18 (RPL5), which are both critical for producing ribosomal subunits and for stabilizing the p53 tumor suppressor when ribosome biogenesis is compromised. Expression of HEATR3 variants or repression of HEATR3 expression in primary cells, cell lines of various origins, and yeast models impairs growth, differentiation, pre-ribosomal RNA processing, and ribosomal subunit formation reminiscent of DBA models of large subunit RP gene variants. Consistent with a role of HEATR3 in RP import, HEATR3-depleted cells or patient-derived fibroblasts display reduced nuclear accumulation of uL18. Hematopoietic progenitor cells expressing HEATR3 variants or small-hairpin RNAs knocking down HEATR3 synthesis reveal abnormal acceleration of erythrocyte maturation coupled to severe proliferation defects that are independent of p53 activation. Our study uncovers a new pathophysiological mechanism leading to DBA driven by biallelic HEATR3 variants and the destabilization of a nuclear import protein important for ribosome biogenesis.

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells.

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10-R98S). Our data support that RPL10-R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock-in of RPL10-R98S was associated with a 1.7- to 2.5-fold increased production of four transiently expressed recombinant proteins and 1.7-fold for one out of two stably expressed proteins. In CHO-S cells, eGFP reached a 2-fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10-R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10-R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

Repurposing the Antidepressant Sertraline as SHMT Inhibitor to Suppress Serine/Glycine Synthesis-Addicted Breast Tumor Growth.

Metabolic rewiring is a hallmark of cancer that supports tumor growth, survival, and chemotherapy resistance. Although normal cells often rely on extracellular serine and glycine supply, a significant subset of cancers becomes addicted to intracellular serine/glycine synthesis, offering an attractive drug target. Previously developed inhibitors of serine/glycine synthesis enzymes did not reach clinical trials due to unfavorable pharmacokinetic profiles, implying that further efforts to identify clinically applicable drugs targeting this pathway are required. In this study, we aimed to develop therapies that can rapidly enter the clinical practice by focusing on drug repurposing, as their safety and cost-effectiveness have been optimized before. Using a yeast model system, we repurposed two compounds, sertraline and thimerosal, for their selective toxicity against serine/glycine synthesis-addicted breast cancer and T-cell acute lymphoblastic leukemia cell lines. Isotope tracer metabolomics, computational docking, enzymatic assays, and drug-target interaction studies revealed that sertraline and thimerosal inhibit serine/glycine synthesis enzymes serine hydroxymethyltransferase and phosphoglycerate dehydrogenase, respectively. In addition, we demonstrated that sertraline's antiproliferative activity was further aggravated by mitochondrial inhibitors, such as the antimalarial artemether, by causing G1-S cell-cycle arrest. Most notably, this combination also resulted in serine-selective antitumor activity in breast cancer mouse xenografts. Collectively, this study provides molecular insights into the repurposed mode-of-action of the antidepressant sertraline and allows to delineate a hitherto unidentified group of cancers being particularly sensitive to treatment with sertraline. Furthermore, we highlight the simultaneous inhibition of serine/glycine synthesis and mitochondrial metabolism as a novel treatment strategy for serine/glycine synthesis-addicted cancers.

Hallmarks of ribosomopathies.

Ribosomopathies are diseases caused by defects in ribosomal constituents or in factors with a role in ribosome assembly. Intriguingly, congenital ribosomopathies display a paradoxical transition from early symptoms due to cellular hypo-proliferation to an elevated cancer risk later in life. Another association between ribosome defects and cancer came into view after the recent discovery of somatic mutations in ribosomal proteins and rDNA copy number changes in a variety of tumor types, giving rise to somatic ribosomopathies. Despite these clear connections between ribosome defects and cancer, the molecular mechanisms by which defects in this essential cellular machinery are oncogenic only start to emerge. In this review, the impact of ribosomal defects on the cellular function and their mechanisms of promoting oncogenesis are described. In particular, we discuss the emerging hallmarks of ribosomopathies such as the appearance of 'onco-ribosomes' that are specialized in translating oncoproteins, dysregulation of translation-independent extra-ribosomal functions of ribosomal proteins, rewired cellular protein and energy metabolism, and extensive oxidative stress leading to DNA damage. We end by integrating these findings in a model that can provide an explanation how ribosomopathies could lead to the transition from hypo- to hyper-proliferation in bone marrow failure syndromes with elevated cancer risk.

Translatome analysis reveals altered serine and glycine metabolism in T-cell acute lymphoblastic leukemia cells.

Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH.

Correction: The ribosomal RPL10 R98S mutation drives IRES-dependent BCL-2 translation in T-ALL.

Following the publication of this article, the authors noted that Dr Laura Fancello was not listed among the authors. The corrected author list is given below. Additionally, the following was not included in the author contribution statement: 'L.F. analyzed RNA sequencing data'.

The ribosomal RPL10 R98S mutation drives IRES-dependent BCL-2 translation in T-ALL.

The R98S mutation in ribosomal protein L10 (RPL10 R98S) affects 8% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) cases, and was previously described to impair cellular proliferation. The current study reveals that RPL10 R98S cells accumulate reactive oxygen species which promotes mitochondrial dysfunction and reduced ATP levels, causing the proliferation defect. RPL10 R98S mutant leukemia cells can survive high oxidative stress levels via a specific increase of IRES-mediated translation of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2), mediating BCL-2 protein overexpression. RPL10 R98S selective sensitivity to the clinically available Bcl-2 inhibitor Venetoclax (ABT-199) was supported by suppression of splenomegaly and the absence of human leukemia cells in the blood of T-ALL xenografted mice. These results shed new light on the oncogenic function of ribosomal mutations in cancer, provide a novel mechanism for BCL-2 upregulation in leukemia, and highlight BCL-2 inhibition as a novel therapeutic opportunity in RPL10 R98S defective T-ALL.

Ribosomal Lesions Promote Oncogenic Mutagenesis.

Ribosomopathies are congenital disorders caused by mutations in ribosomal proteins (RP) or assembly factors and are characterized by cellular hypoproliferation at an early stage. Paradoxically, many of these disorders have an elevated risk to progress to hyperproliferative cancer at a later stage. In addition, somatic RP mutations have recently been identified in various cancer types, for example, the recurrent RPL10-R98S mutation in T-cell acute lymphoblastic leukemia (T-ALL) and RPS15 mutations in chronic lymphocytic leukemia (CLL). We previously showed that RPL10-R98S promotes expression of oncogenes, but also induces a proliferative defect due to elevated oxidative stress. In this study, we demonstrate that this proliferation defect is eventually rescued by RPL10-R98S mouse lymphoid cells that acquire 5-fold more secondary mutations than RPL10-WT cells. The presence of RPL10-R98S and other RP mutations also correlated with a higher mutational load in patients with T-ALL, with an enrichment in NOTCH1-activating lesions. RPL10-R98S-associated cellular oxidative stress promoted DNA damage and impaired cell growth. Expression of NOTCH1 eliminated these phenotypes in RPL10-R98S cells, in part via downregulation of PKC-θ, with no effect on RPL10-WT cells. Patients with RP-mutant CLL also demonstrated a higher mutational burden, enriched for mutations that may diminish oxidative stress. We propose that oxidative stress due to ribosome dysfunction causes hypoproliferation and cellular insufficiency in ribosomopathies and RP-mutant cancer. This drives surviving cells, potentiated by genomic instability, to acquire rescuing mutations, which ultimately promote transition to hyperproliferation. SIGNIFICANCE: Ribosomal lesions cause oxidative stress and increase mutagenesis, promoting acquisition of rescuing mutations that stimulate proliferation.