Abstract Ribosome biogenesis (Ribi) is essential to cellular function and often deregulated in cancer, making it an attractive therapeutic target. AML cells are known to have enlarged nuclei suggesting increased Ribi is needed to fuel AML growth. Hundreds of Ribi factors are crucial for rRNA processing and ribosome assembly. However, the role of Ribi factors in AML is poorly understood. To address this, we analysed proteomic profiles of 55 poor-risk AML patients and 4 normal myeloid samples. We identified 759 downregulated and 1646 upregulated proteins. Strikingly, rRNA processing is the most enriched pathway in the upregulated proteins, followed by cell cycle. Furthermore, rRNA processing, synthesis and ribosome subunit maturation pathways are increased in most AML molecular subtypes but not in some others including IDH1/2, SRSF2 and NPM1 mutants. To understand how upregulated rRNA processing factors benefit AML, we focused on MYBBP1A (Fold change=2.3; FDR=1.11x10-11) since it was also one of the top hits in our previous in vivo shRNA dropout screen in leukemic stem cells. Interestingly, MYBBP1A protein and RNA levels in AML patients are not correlated, suggesting MYBBP1A upregulation is post-transcriptional. MYBBP1A is mainly a co-transcription factor suppressing tumor in several solid cancers, and recently in MLL-ENL AML though with an unclear mechanism. Thus, MYBBP1A's role in AML is controversial and its molecular function in this context is unclear. Immunoblot verified increased MYBBP1A protein levels in primary AML patients and 11 AML cell lines compared to normal cord blood hematopoietic stem/progenitor cells (HSPCs) and bone marrow cells. Knockdown (KD) of MYBBP1A in several human AML cell lines using RNAi and CRISPR-Cas9 approaches inhibited cell proliferation, elevated apoptosis and myeloid differentiation. Survival of recipient mice transplanted with MYBBP1A depleted AML cells (MOLM13) was significantly improved compared to the control animals. Additionally, Mybbp1a knockout (KO) in MLL-AF9 Mybbp1afl/fl puroCreER murine models delayed leukemogenesis (median survival 32 days in wildtype vs. 137 days in Mybbp1a KO with 2 mice not-reached), suggesting its requirement for leukemia maintenance. Interestingly, MYBBP1A KD is also detrimental to both human and mouse NPM1 mutant AMLs (OCI-AML3 and Npm1c/Flt3ITD murine AML model). Using co-culture system with stromal cells, we show that MYBBP1A KD suppresses primary AML patient cell growth. Importantly, MYBBP1A KD in CD34+ HSPCs decreased their clonogenicity by 50%, with no effects on apoptosis, suggesting a therapeutic index. Mechanistically, our co-immunoprecipitation and imaging analysis shows MYBBP1A localises in nucleolus and interacts with multiple ribosomal proteins and Ribi factors including fibrillarin (FBL). MYBBP1A KD impaired FBL recruitment into nucleolus, without changing total FBL protein level. Nascent RNA capture revealed that rRNA synthesis and processing was modulated by MYBBP1A KD as evident by reduction in 47S, 5.8S, 18S and 28S rRNA. To understand how this affects protein synthesis, we performed polysome profiling and found that polysome:monosome-subunit ratio is significantly reduced upon MYBBP1A KD, suggesting global translation is modulated. This is consistent with our OP-Puro incorporation analysis. To understand MYBBP1A-regulated pathways, we performed proteomic analysis and unexpectedly found only a small subset of genes significantly changed at protein level, including all 6 components of the DNA unwinding complex MCM2-7. To assess if MYBBP1A may also act as a transcription factor at these loci, we carried out ChIP-seq analysis and revealed that MYBBP1A binds MCM promoters. Moreover, MCM6 and 7 mRNAs are reduced upon MYBBP1A loss. Cell cycle analysis shows that MYBBP1A depleted cells are arrested at G1 phase. Notably, these MCM proteins are part of the cell cycle program upregulated in the AML patient proteomics. Expression of shRNA-resistant MYBBP1A fully rescued MCM6 level, proliferation and apoptosis defects of KD cells. These data suggest that MYBBP1A also controls AML cells via regulating MCM gene expression. In summary, our study demonstrated that Ribi factors are aberrantly upregulated and may represent new therapeutic potential in AML. We uncover that the Ribi factor MYBBP1A is required for AML cell growth via playing a dual role as a regulator of ribosome biogenesis and translation, as well as a transcription factor of MCM complex.
To et al. (Mon,) studied this question.