Shihan He

Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

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.

Structural insights into inhibition of the bivalent menin-MLL interaction by small molecules in leukemia

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.

High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein–Protein Interaction

The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ∼288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin–MLL interaction with IC50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.

The same site on the integrase-binding domain of lens epithelium-derived growth factor is a therapeutic target for MLL leukemia and HIV.

Lens epithelium-derived growth factor (LEDGF) is a chromatin-associated protein implicated in leukemia and HIV type 1 infection. LEDGF associates with mixed-lineage leukemia (MLL) fusion proteins and menin and is required for leukemic transformation. To better understand the molecular mechanism underlying the LEDGF integrase-binding domain (IBD) interaction with MLL fusion proteins in leukemia, we determined the solution structure of the MLL-IBD complex. We found a novel MLL motif, integrase domain binding motif 2 (IBM2), which binds to a well-defined site on IBD. Point mutations within IBM2 abolished leukemogenic transformation by MLL-AF9, validating that this newly identified motif is essential for the oncogenic activity of MLL fusion proteins. Interestingly, the IBM2 binding site on IBD overlaps with the binding site for the HIV integrase (IN), and IN was capable of efficiently sequestering IBD from the menin-MLL complex. A short IBM2 peptide binds to IBD directly and inhibits both the IBD-MLL/menin and IBD-IN interactions. Our findings show that the same site on IBD is involved in binding to MLL and HIV-IN, revealing an attractive approach to simultaneously target LEDGF in leukemia and HIV.

Menin-MLL inhibitors block oncogenic transformation by MLL fusion proteins in a fusion partner independent manner

Menin-MLL inhibitors block oncogenic transformation by MLL-fusion proteins in a fusion partner-independent manner

BMI1 regulates PRC1 architecture and activity through homo- and hetero-oligomerization

BMI1 is a core component of the polycomb repressive complex 1 (PRC1) and emerging data support a role of BMI1 in cancer. The central domain of BMI1 is involved in protein–protein interactions and is essential for its oncogenic activity. Here, we present the structure of BMI1 bound to the polyhomeotic protein PHC2 illustrating that the central domain of BMI1 adopts an ubiquitin-like (UBL) fold and binds PHC2 in a β-hairpin conformation. Unexpectedly, we find that the UBL domain is involved in homo-oligomerization of BMI1. We demonstrate that both the interaction of BMI1 with polyhomeotic proteins and homo-oligomerization via UBL domain are necessary for H2A ubiquitination activity of PRC1 and for clonogenic potential of U2OS cells. Here, we also emphasize need for joint application of NMR spectroscopy and X-ray crystallography to determine the overall structure of the BMI1–PHC2 complex.

Abstract 3520: Small molecule inhibitors of ring1B-Bmi1 E3 ligase target polycomb repressive complex 1 activity and regulate cell proliferation

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Emerging evidence implicates the role of Ring1B and Bmi1 in the maintenance of embryonic stem cells and regulating adult stem cells self-renewal. Both Ring1B and Bmi1 are part of Poly-comb Repressive Complex 1(PRC1) and execute gene silencing through histone H2A lysine119 ubiquitination (UbH2A K119). In addition, Bmi1 is also required for the self-renewal capacity of leukemic stem cells, suggesting that Poly-comb Group proteins may contribute to oncogenic transformation by regulating stem cell self-renewal. Targeting cancer stem cell self-renewal has been proposed as a therapeutic goal and could be an effective approach to control tumor growth. In this study, we report the development and characterization of small molecule inhibitors against Ring1B-Bmi1 complex which interfere with its E3 ligase function. We developed ligands binding to Ring1B-Bmi1 employing NMR fragment-based screening. We further performed extensive medicinal chemistry optimization and synthesized over 80 analogs of the initial fragment hit and improved its binding affinity to Ring1B-Bmi1 complex by more than 1000-fold. The activity of developed small molecule inhibitors was analyzed by cellular and biochemical studies including in vitro ubiquitination assay and quantitative analysis of p16 expression. Mechanistically, we found that our most potent compounds inhibit E3 ligase activity of Ring1B-Bmi1 at low micromolar concentrations. As expected, treatment of HeLa and leukemic cell lines with these compounds demonstrated reduced UbH2A K119 levels and increased expression of p16. In addition, presence of the lead compounds also reduces leukemic cell proliferation as determined by MTT assays. Furthermore, the comparison between knockdown effects of Ring1B/Bmi1 with small molecule inhibitors revealed on target effect of the lead compounds. To our knowledge, these compounds represent the first small molecule inhibitors that directly bind to Ring1B-Bmi1 and inhibit its E3 ubiquitin ligase activity and have a strong potential for further development into potent anticancer agents. Citation Format: Shirish Shukla, Qingjie Zhao, Weijang Ying, Felicia Gray, Kelly Vandenberg, George Lund, Bohdan Boytsov, Shihan He, Jolanta Grembecka, Tomasz Cierpicki. Small molecule inhibitors of ring1B-Bmi1 E3 ligase target polycomb repressive complex 1 activity and regulate cell proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2015-3520

Abstract 2534: High-affinity small molecule inhibitors of the menin-MLL interaction reverse oncogenic transformation mediated by MLL fusion proteins in leukemia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The protein-protein interaction (PPI) between menin and Mixed Lineage Leukemia (MLL) plays a critical role in acute leukemias with translocations of MLL gene, and inhibition of this interaction with small molecules represents a new potential therapeutic strategy for the MLL leukemia patients. We identified novel small molecule inhibitors of the menin-MLL interaction with distinct molecular scaffolds by performing a High Throughput Screening (HTS) of over 300,000 compounds. Extensive medicinal chemistry efforts performed for two lead classes to improve inhibitory activity of these compounds resulted in menin-MLL inhibitors with low nanomolar binding affinities. The menin-inhibitor co-crystal structures revealed that these compounds directly bind to menin and closely mimic the key interactions of MLL with menin, resulting in their high binding affinity. Interestingly, the hydroxymethylpiperidine class of the menin-MLL inhibitors extends beyond the MLL binding region on menin, providing additional opportunity for their optimization. We combined extensive crystallography studies with structure-based design to perform rational optimization and scaffold modification to rapidly improve activity and modulate physicochemical properties of the menin-MLL inhibitors. Treatment of MLL leukemia cells with the most potent menin-MLL inhibitors we developed resulted in very effective and selective inhibition of cell proliferation, induced apoptosis and differentiation of these cells. These effects were associated with downregulation of Hoxa9 and Meis1 expression, the downstream targets of MLL fusion proteins required for their leukemogenicity, demonstrating a very specific mechanism of action for these newly developed menin-MLL inhibitors. In vivo studies are currently undergoing to assess the effect of these compounds on leukemia progression in animal models of MLL leukemia. Our studies provide a novel and very attractive scaffolds for further development as a new potential therapeutic approach for the MLL leukemia patients. Citation Format: Jolanta E. Grembecka, Shihan He, Timothy J. Senter, Dmitry Borkin, Jonathan Pollock, Changho Han, Sunil Kumar Upadhyay, Trupta Purohit, Hongzhi Miao, Rocco D. Gogliotti D. Gogliotti, Craig W. Lindsley, Tomasz Cierpicki, Shaun R. Stauffer. High-affinity small molecule inhibitors of the menin-MLL interaction reverse oncogenic transformation mediated by MLL fusion proteins in leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2534. doi:10.1158/1538-7445.AM2014-2534

Abstract 3225: LEDGF IBD domain represents therapeutic target for MLL leukemia and HIV

Lens epithelium-derived growth factor (LEDGF) is a chromatin associated protein implicated in cell survival, cancer, autoimmune diseases and HIV pathogenesis. LEDGF is also involved in recurring chromosomal translocations with nucleoporin NUP98 in acute leukemia, and the LEDGF/p75 isoform is consistently upregulated in a subset of acute myeloid leukemias (AML) resistant to chemotherapy. Moreover, it has been established that LEDGF is an essential co-factor required for oncogenic activity of MLL fusion proteins in leukemia. LEDGF interacts simultaneously with MLL and menin and is necessary for up-regulation of HOXA9 gene and for leukemogenic transformation by MLL fusion proteins in vivo. Therefore, LEDGF represent a valuable molecular target for therapeutic intervention as a novel targeted therapy for MLL leukemia patients. Furthermore, targeting LEDGF may represent an attractive alternative to inhibition of the menin-MLL interaction as it will allow preserving the function of menin, a known tumor suppressor in endocrine tissues. We determined the solution structure of MLL fragment bound to LEDGF IBD domain and identified a novel hydrophobic motif within MLL, which we called IBM2, that is required for high affinity interaction with LEDGF. Point mutations within IBM2 abolished leukemogenic activity of MLL-AF9, indicating that IBM2 represents a critical site for formation of a high affinity menin-MLL-LEDGF complex. Furthermore, we found that short peptide corresponding to IBM2 binds to IBD domain and disrupts the interaction of LEDGF with the menin-MLL complex, providing a proof of concept that the novel interface on IBD domain we identified represents a druggable site for small molecule intervention. LEDGF plays also an essential role in pathogenesis of HIV-1 virus and is required for integration of viral cDNA. Targeting HIV integrase to disrupt the interaction with LEDGF has been recognized as an attractive approach to develop new anti-viral agents. Importantly, the new MLL binding site on IBD domain we found overlaps with the binding site of HIV integrase. Therefore, targeting the IBM2 site on IBD may represent a novel approach to target the LEDGF-integrase interaction. In summary, our findings pave the way towards development of new therapeutic agents with dual applications for both MLL leukemias and HIV. Citation Format: Tomasz Cierpicki, Marcelo J. Murai, Jonathan Pollock, Trupta Purohit, Shihan He, Adam Yokom, Jay L. Hess, Andrew G. Muntean, Jolanta Grembecka. LEDGF IBD domain represents therapeutic target for MLL leukemia and HIV. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3225. doi:10.1158/1538-7445.AM2014-3225