N6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA that is required for the differentiation of mouse embryonic stem cells. However, it remains unknown whether m6A controls differentiation of normal and/or malignant myeloid hematopoietic cells. Here we show that shRNA-mediated depletion of the m6A-forming enzyme METTL3 in human hematopoietic stem/progenitor cells promotes differentiation coupled with reduced proliferation. Conversely, overexpression of wild-type METTL3, but not the catalytic-dead form of METTL3, inhibits differentiation and increases cell growth. METTL3 mRNA and protein is expressed more abundantly in acute myeloid leukemia (AML) cells compared to healthy hematopoietic stem/progenitor cells and other types of tumors. Furthermore, METTL3 depletion in humanmyeloid leukemia cell lines induces differentiation and apoptosis and delays leukemia in recipient mice in vivo. Single-nucleotide resolution mapping of m6A coupled with ribosome profiling reveals that m6A promotes the translation of c-MYC, BCL2 and PTEN mRNAs in human myeloid leukemia MOLM13 cells. Moreover, loss of METTL3 leads to increased levels of pAKT, which contributes to the differentiation effects of METTL3 depletion. Overall these results provide a rationale for therapeutic targeting of METTL3 in myeloid leukemia.

SUMMARY Myeloid malignancy is increasingly viewed as a disease spectrum, comprising hematopoietic disorders that extend across a phenotypic continuum ranging from clonal hematopoiesis to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we derived a collection of iPSC lines capturing a range of disease stages encompassing preleukemia, low-risk MDS, high-risk MDS and secondary AML. Upon differentiation, we found hematopoietic phenotypes of graded severity and/or stage specificity that together delineate a phenotypic roadmap of disease progression culminating in serially transplantable leukemia. We also show that disease stage transitions, both reversal and progression, can be modeled in this system using genetic correction or introduction of mutations via CRISPR/Cas9, and that this iPSC-based approach can be used to uncover disease stage-specific responses to drugs. Our study therefore provides insight into the cellular events demarcating the initiation and progression of myeloid transformation and a new platform for testing genetic and pharmacological interventions.

Recent evidence suggests that transcription initiation by all three eukaryotic RNA polymerases involves a complex of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here, we map the functional domains of the nucleolar HMG box protein hUBF, which binds to the human rRNA promoter and stimulates transcription by RNA polymerase I through cooperative interactions with a distinct TBP-TAF complex, hSL1. DNase I footprint analysis of mutant hUBF proteins and of a synthetic peptide of 84 amino acids reveals that HMG box 1 is necessary and sufficient for DNA sequence specificity, whereas other HMG boxes and the amino terminus modulate the binding efficiency, hUBF contains multiple activation domains that include the acidic carboxyl terminus and three HMG boxes. HMG boxes 3 and 4 and the acidic tail contribute significantly to an extended footprinting pattern in the presence of hSL1, suggestive of specific protein-protein interactions. Moreover, the inability of xUBF from Xenopus laevis to form an initiation complex with hSL1 can be overcome by hybrid proteins containing human HMG box 4 and the acidic carboxyl terminus. These results strongly suggest an important role of transcription activation domains of hUBF in mediating interactions with the TBP-TAF complex hSL1. In recent years considerable progress has been made toward elucidating the molecular mechanisms of transcription initiation. In particular, a multitude of sequence-specific transcription factors that bind to cis-acting promoter and enhancer elements have been identified and their structure and function have been analyzed (Johnson and McKnight 1989;Mitchell and Tjian 1989). Moreover, a number of components of the basal transcriptional apparatus have been implicated as targets for activation (Sawadogo and Sentenac 1990;Greenblatt 1991;Roeder 1991; Pugh and Tjian 1992). Although domains involved in transcriptional activation by sequence-specific transcription factors have been identified, their targets in the basal transcriptional machinery remained elusive.The recent identification of coactivators and TATAbinding protein-associated factors (TAFs) that are associated with the TATA-binding protein (TBP) in the TFIID fraction of the RNA polymerase II (Pol II) system focused our attention on a novel class of transcription factors. At least some of these TAFs are required for activation by sequence-specific regulatory proteins Tjian 1990, 1991;Dynlacht et al. 1991;Tanese et al. 1991;Gill and Tjian 1992). Thus, there appear to be adaptor molecules that link the basal transcriptional apparatus to the activators. Several recent reports indicate that TBP is not only involved in transcription initiation by RNA Pol II but is also necessary for transcription by RNA polymerase III (Pol III) as well as RNA polymerase I (Pol I) (Lobo et al. 1991;Margottin et al. 1991;Simmen et al. 1991;Comai et al. 1992;Cormack and Struhl 1992;Schultz et al. 1992;White et al. 1992). These studies strongly support the unifying principle that in spite of vast differenc...

The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08–2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI’s oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.

The M APK/ E RK K inase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology 1 , and more recently in immunotherapy 2 , 3 and aging 4 . However, many MEK inhibitors (MEKi) are limited due to on-target toxicities 5 – 7 and drug resistance 8 – 10 . Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR ( K inase S uppressor of R as) with various MEKi, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. Through complexation, KSR remodels the prototypical MEKi allosteric pocket thereby impacting binding and kinetics, including drug residence time. Moreover, trametinib binds KSR-MEK but disrupts the related RAF-MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these subcomplexes. Based on these insights we created trametiglue, which limits adaptive resistance to MEKi through enhanced interfacial binding. Together, our results reveal the plasticity of an interface pocket within MEK subcomplexes that has implications for the design of next generation drugs targeting the RAS pathway.