Functional diversity of inhibitors tackling the differentiation blockage of MLL-rearranged leukemia.
ABSTRACT: INTRODUCTION:The chromosomal rearrangements of the mixed-lineage leukemia gene MLL (KMT2A) have been extensively characterized as a potent oncogenic driver in leukemia. For its oncogenic function, most MLL-fusion proteins exploit the multienzyme super elongation complex leading to elevated expression of MLL target genes. High expression of MLL target genes overwrites the normal hematopoietic differentiation program, resulting in undifferentiated blasts characterized by the capacity to self-renew. Although extensive resources devoted to increased understanding of therapeutic targets to overcome de-differentiation in ALL/AML, the inter-dependencies of targets are still not well described. The majority of inhibitors potentially interfering with MLL-fusion protein driven transformation have been characterized in individual studies, which so far hindered their direct cross-comparison. METHODS:In our study, we characterized head-to-head clinical stage inhibitors for BET, DHODH, DOT1L as well as two novel inhibitors for CDK9 and the Menin-MLL interaction with a focus on differentiation induction. We profiled those inhibitors for global gene expression effects in a large cell line panel and examined cellular responses such as inhibition of proliferation, apoptosis induction, cell cycle arrest, surface marker expression, morphological phenotype changes, and phagocytosis as functional differentiation readout. We also verified the combination potential of those inhibitors on proliferation and differentiation level. RESULTS:Our analysis revealed significant differences in differentiation induction and in modulating MLL-fusion target gene expression. We observed Menin-MLL and DOT1L inhibitors act very specifically on MLL-fused leukemia cell lines, whereas inhibitors of BET, DHODH and P-TEFb have strong effects beyond MLL-fusions. Significant differentiation effects were detected for Menin-MLL, DOT1L, and DHODH inhibitors, whereas BET and CDK9 inhibitors primarily induced apoptosis in AML/ALL cancer models. For the first time, we explored combination potential of the abovementioned inhibitors with regards to overcoming the differentiation blockage. CONCLUSION:Our findings show substantial diversity in the molecular activities of those inhibitors and provide valuable insights into the further developmental potential as single agents or in combinations in MLL-fused leukemia.
Project description:Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.
Project description:Inhibition of the menin-mixed lineage leukemia (MLL) protein-protein interaction is a promising new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). We describe herein our structure-based design, synthesis, and evaluation of a new class of small-molecule inhibitors of the menin-MLL interaction (hereafter called menin inhibitors). Our efforts have resulted in the discovery of highly potent menin inhibitors, as exemplified by compound 42 (M-89). M-89 binds to menin with a Kd value of 1.4 nM and effectively engages cellular menin protein at low nanomolar concentrations. M-89 inhibits cell growth in the MV4;11 and MOLM-13 leukemia cell lines carrying MLL fusion with IC50 values of 25 and 55 nM, respectively, and demonstrates >100-fold selectivity over the HL-60 leukemia cell line lacking MLL fusion. The determination of a co-crystal structure of M-89 in a complex with menin provides the structural basis for their high-affinity interaction. Further optimization of M-89 may lead to a new class of therapy for the treatment of MLL leukemia.
Project description:Menin functions as a critical oncogenic cofactor of mixed lineage leukemia (MLL) fusion proteins in the development of acute leukemias, and inhibition of the menin interaction with MLL fusion proteins represents a very promising strategy to reverse their oncogenic activity. MLL interacts with menin in a bivalent mode involving 2 N-terminal fragments of MLL. In the present study, we reveal the first high-resolution crystal structure of human menin in complex with a small-molecule inhibitor of the menin-MLL interaction, MI-2. The structure shows that the compound binds to the MLL pocket in menin and mimics the key interactions of MLL with menin. Based on the menin-MI-2 structure, we developed MI-2-2, a compound that binds to menin with low nanomolar affinity (K(d) = 22nM) and very effectively disrupts the bivalent protein-protein interaction between menin and MLL. MI-2-2 demonstrated specific and very pronounced activity in MLL leukemia cells, including inhibition of cell proliferation, down-regulation of Hoxa9 expression, and differentiation. Our results provide the rational and essential structural basis to design next generation of inhibitors for effective targeting of the menin-MLL interaction in leukemia and demonstrate a proof of concept that inhibition of complex multivalent protein-protein interactions can be achieved by a small-molecule inhibitor.
Project description:Trithorax and polycomb group proteins antagonistically regulate the transcription of many genes, and cancer can result from the disruption of this regulation. Deregulation of trithorax function occurs through chromosomal translocations involving the trithorax gene MLL, leading to the expression of MLL fusion proteins and acute leukemia. It is poorly understood how MLL fusion proteins block differentiation, a hallmark of leukemogenesis. We analyzed the effect of acute depletion of menin, a close partner of MLL that is critical for MLL and MLL-AF9 recruitment to target genes, on MLL-AF9 leukemia cell differentiation using an in vivo model. We performed cDNA microarray analysis of menin-regulated genes from primary leukemia cells to determine menin-regulated pathways involved in suppressing MLL-AF9 leukemia cell differentiation. We found that menin binds the promoter of the polycomb gene Ezh2, and promotes its expression. EZH2 interacts with the differentiation-promoting transcription factor C/EBPα and represses C/EBPα target genes. Menin depletion reduces MLL binding to the Ezh2 locus, EZH2 expression, and EZH2 binding and repressive H3K27 methylation at C/EBPα target genes, thereby inducing the expression of pro-differentiation C/EBPα targets. In conclusion, our results show that in contrast to its classical role antagonizing trithorax function, the polycomb group protein EZH2 collaborates with trithorax-associated menin to block MLL-AF9 leukemia cell differentiation, uncovering a novel mechanism for suppression of C/EBPα and leukemia cell differentiation, through menin-mediated upregulation of EZH2.
Project description:The aim of this study was to better understand how mixed lineage leukemia (MLL) fusion proteins deregulate the expression of genes critical for leukemia.The transforming domain of one of the most common MLL fusion partners, AF9, was immunopurified after expression in myeloblastic M1 cells, and associating proteins were identified by mass spectrometric analysis. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was used to determine how binding of associating proteins compare across Hoxa9 and Meis1 in cell lines with and without MLL fusion proteins and how binding is altered during gene down-regulation and differentiation.Consistent with earlier purifications of ENL and AF4 from 293 cells, the 90 amino acid C-terminal domain of AF9 associates with many other MLL translocation partners including Enl, Af4, Laf4, Af5q31, Ell, and Af10. This complex, termed elongation assisting proteins (EAPs), also contains the RNA polymerase II C-terminal domain kinase Cdk9/Cyclin T1/T2 (pTEFb) and the histone H3 lysine 79 methyltransferase Dot1L. Myeloid cells transformed by MLL fusions show higher levels and a broader distribution of EAP components at genes critical for leukemia. Inhibition of EAP components pTEFb and Dot1l show that both contribute significantly to activation of Hoxa9 and Meis1 expression. EAP is dynamically associated with the Hoxa9 and Meis1 loci in hematopoietic cells and rapidly dissociates during induction of differentiation. In the presence of MLL fusion proteins, its dissociation is prevented.The findings suggest that MLL fusion proteins deregulate genes critical for leukemia by excessive recruitment and impaired dissociation of EAP from target loci.
Project description:Chromosomal translocations targeting the mixed lineage leukemia (MLL) gene result in MLL fusion proteins that are found in aggressive human acute leukemias. Disruption of MLL by such translocations leads to overexpression of Hox genes, resulting in a blockage of hematopoietic differentiation that ultimately leads to leukemia. Menin, which directly binds MLL, has been identified as an essential oncogenic co-factor required for the leukemogenic activity of MLL fusion proteins. Here, we characterize the molecular basis of the MLL-menin interaction. Using (13)C-detected NMR experiments, we have mapped the residues within the intrinsically unstructured fragment of MLL that are required for binding to menin. Interestingly, we found that MLL interacts with menin with a nanomolar affinity (K(d) ? 10 nM) through two motifs, MBM1 and MBM2 (menin binding motifs 1 and 2). These motifs are located within the N-terminal 43-amino acid fragment of MLL, and the MBM1 represents a high affinity binding motif. Using alanine scanning mutagenesis of MBM1, we found that the hydrophobic residues Phe(9), Pro(10), and Pro(13) are most critical for binding. Furthermore, based on exchange-transferred nuclear Overhauser effect measurements, we established that MBM1 binds to menin in an extended conformation. In a series of competition experiments we showed that a peptide corresponding to MBM1 efficiently dissociates the menin-MLL complex. Altogether, our work establishes the molecular basis of the menin interaction with MLL and MLL fusion proteins and provides the necessary foundation for development of small molecule inhibitors targeting this interaction in leukemias with MLL translocations.
Project description:Understanding the underlying molecular mechanisms of defined cancers is crucial for effective personalized therapies. Translocations of the mixed-lineage leukemia (MLL) gene produce fusion proteins such as MLL-AF4 that disrupt epigenetic pathways and cause poor-prognosis leukemias. Here, we find that at a subset of gene targets, MLL-AF4 binding spreads into the gene body and is associated with the spreading of Menin binding, increased transcription, increased H3K79 methylation (H3K79me2/3), a disruption of normal H3K36me3 patterns, and unmethylated CpG regions in the gene body. Compared to other H3K79me2/3 marked genes, MLL-AF4 spreading gene expression is downregulated by inhibitors of the H3K79 methyltransferase DOT1L. This sensitivity mediates synergistic interactions with additional targeted drug treatments. Therefore, epigenetic spreading and enhanced susceptibility to epidrugs provides a potential marker for better understanding combination therapies in humans.
Project description:Rearrangements of MLL (encoding lysine-specific methyltransferase 2A and officially known as KMT2A; herein referred to as MLL to denote the gene associated with mixed-lineage leukemia) generate MLL fusion proteins that bind DNA and drive leukemogenic gene expression. This gene expression program is dependent on the disruptor of telomeric silencing 1-like histone 3 lysine 79 (H3K79) methyltransferase DOT1L, and small-molecule DOT1L inhibitors show promise as therapeutics for these leukemias. However, the mechanisms underlying this dependency are unclear. We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL fusion target genes after DOT1L inhibition. DOT1L inhibits chromatin localization of a repressive complex composed of SIRT1 and the H3K9 methyltransferase SUV39H1, thereby maintaining an open chromatin state with elevated H3K9 acetylation and minimal H3K9 methylation at MLL fusion target genes. Furthermore, the combination of SIRT1 activators and DOT1L inhibitors shows enhanced antiproliferative activity against MLL-rearranged leukemia cells. These results indicate that the dynamic interplay between chromatin regulators controlling the activation and repression of gene expression could provide novel opportunities for combination therapy.
Project description:DOT1L is a unique histone methyltransferase that targets the histone H3 lysine 79 (H3K79) residue for mono-, di- and tri- methylation. Histone H3K79 mono- and di-methylation results in active gene transcription, while H3K79 tri-methylation is associated with gene repression. DOT1L has a critical role in regulating gene transcription, development, cell cycle progression, somatic reprogramming and DNA damage repair. DOT1L interacts with Mixed Lineage Leukemia (MLL) fusion proteins, leading to enhanced H3K79 methylation, maintenance of open chromatin, overexpression of downstream oncogenes and leukemogenesis. Importantly, small molecule DOT1L inhibitors have been recently developed, and one of the DOT1L inhibitors is already under investigation in a Phase I clinical trial in patients with MLL fusion gene-driven leukemia.
Project description:Chromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification.