Nucleotide depletion reveals the impaired ribosomebiogenesis checkpoint as a barrier against DNA damage

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21‐mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC‐mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability

IRBC-induced p53-dependent Cell Death in c-MYC-driven tumors mediated by loss of MCL1

[eng] The oncogene MYC is altered and its expression is deregulated in up to 70% of human cancers, including B cell neoplasms. Earlier studies in a mouse model of Eμ-MYC-driven B-cell lymphomas reported that oncogenic MYC relies on aberrant rates of ribosome biogenesis (RiBi) and protein synthesis to sustain rapid growth and proliferation of B-cell lymphomas. As MYC driven B-cell lymphomas are addicted to hyperactivation of RiBi, it has emerged as a potential clinical target. However, it is unclear whether targeting RiBi induces regression of MYC-driven tumors by decreasing translational capacity and/or by inducing the impaired ribosome biogenesis checkpoint (IRBC), leading to p53 stabilization. We set to address this question by generating an inducible system in Eμ-MYC-driven lymphoma cells to deplete either one of two essential 60S ribosomal proteins (RPs), RPL7a or RPL11, the latter a component of the IRBC complex. Depletion of either RP mRNA by ~50% had an equivalent impact on RiBi, protein synthesis and cell growth, however only depletion of RPL7a led to the induction of the IRBC, p53 stabilization, and acute induction of apoptosis. Importantly, we observed that this response is driven by the selective degradation of the antiapoptotic form of MCL1, of the BCL2 family, whose overexpression is critical to sustain survival and growth of Eμ-MYC lymphomas. MCL1 is commonly overexpressed in many human cancers, especially in B cell malignancies, is frequently found co-amplified with MYC, and its overexpression is associated with bad prognosis, resistance to therapy and relapse. Despite the tremendous investment in the development of selective MCL1 inhibitors in the clinic, we show that nanomolar concentrations Actinomycin D (ActD), an FDA approved drug for particular types of cancer, specifically disrupts the synthesis of rRNA and RiBi, leading to IRBC activation, p53 stabilization and degradation of the antiapoptotic form of MCL1, and killing Trp53+/+, but not Trp53-/- Eμ-MYC lymphoma cells. Finally, we provide preclinical data that mice bearing Trp53+/+, but not Trp53-/-, Eμ-MYC lymphomas are exquisitely protected from lymphomagenesis by ActD. Therefore, in MYC-driven tumors, the IRBC elicits p53-dependent apoptosis, which is mediated by the loss of the antiapoptotic form of MCL1.[spa] La expresión del oncogen MYC está desregulada en hasta un 70% de los cánceres humanos, incluyendo los neoplasmas de células B. Estudios previos en el modelo murino de linfoma de células B Eμ-MYC demostraron que la expresión oncogénica de MYC requiere de tasas de biogénesis de ribosomas (RiBi) y síntesis de proteínas aberrantes para el rápido crecimiento y proliferación de estos tumores y que son adictos a la hiperactivación de la RiBi, convirtiéndose en una potencial diana terapéutica. Sin embargo, si inhibir la RiBi promueve la regresión tumoral mediante la disminución de la capacidad de traducción y/o por la inducción del punto de control de daño en la biogénesis de ribosomas (IRBC) y la consiguiente estabilización de p53, no está claro. Para resolver esta controversia, generamos un sistema inducible que elimina la proteína ribosomal (RP)L7a o la RPL11 en células Eμ-MYC, las dos constituyentes del ribosoma 60S, pero la última esencial del complejo IRBC. Una reducción del 50% en el mRNA de cualquiera de las dos tiene un impacto similar en la RiBi, la síntesis de proteínas y el crecimiento celular; pero sólo la reducción de la RPL7a induce el IRBC, estabiliza p53 e induce apoptosis. Además, esta respuesta se desencadena mediante la degradación selectiva de la forma antiapoptótica de MCL1, cuya sobreexpresión es crucial para la supervivencia y el crecimiento de los linfomas Eμ-MYC. MCL1 se sobreexpresa en muchos tumores, especialmente en los de células B, frecuentemente co- amplifica con MYC y se asocia a peor prognosis, resistencia y recaída. A pesar de la tremenda inversión en el desarrollo de inhibidores selectivos de MCL1 en la clínica, nosotros demostramos que concentraciones nanomolares de Actinomicina D (ActD), fármaco aprobado por la FDA para tratar ciertos tumores, interrumpe selectivamente la síntesis de rRNA y la RiBi, activa el IRBC, estabiliza p53 y degrada específicamente la forma antiapoptótica de MCL1, acabando con las células de linfoma Eμ-MYC Trp53+/+ pero no con aquellas Trp53-/-. Finalmente, proporcionamos datos preclínicos en los que la ActD protege contra la linfomagénesis a ratones transplantados con linfomas Eμ-MYC Trp53+/+ pero no con linfomas Eμ-MYC Trp53-/-. Por tanto, en estos tumores dirigidos por MYC, el IRBC activa apoptosis por p53, la cual requiere de la degradación de la forma antiapoptótica de MCL1