Combination of menin and kinase inhibitors as an effective treatment for leukemia with NUP98 translocations.

Chromosomal translocations of the nucleoporin 98 (NUP98) gene are found in acute myeloid leukemia (AML) patients leading to very poor outcomes. The oncogenic activity of NUP98 fusion proteins is dependent on the interaction between Mixed Lineage Leukemia 1 and menin. NUP98-rearranged (NUP98-r) leukemia cells also rely on specific kinases, including CDK6 and/or FLT3, suggesting that simultaneous targeting of these kinases and menin could overcome limited sensitivity to single agents. Here, we found that combinations of menin inhibitor, MI-3454, with kinase inhibitors targeting either CDK6 (Palbociclib) or FLT3 (Gilteritinib) strongly enhance the anti-leukemic effect of menin inhibition in NUP98-r leukemia models. We found strong synergistic effects of both combinations on cell growth, colony formation and differentiation in patient samples with NUP98 translocations. These combinations also markedly augmented anti-leukemic efficacy of menin inhibitor in Patient Derived Xenograft models of NUP98-r leukemia. Despite inhibiting two unrelated kinases, when Palbociclib or Gilteritinib were combined with the menin inhibitor, they affected similar pathways relevant to leukemogenesis, including cell cycle regulation, cell proliferation and differentiation. This study provides strong rationale for clinical translation of the combination of menin and kinase inhibitors as novel treatments for NUP98-r leukemia, supporting the unexplored combinations of epigenetic drugs with kinase inhibitors.

Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain.

Polycomb repressive complex 1 (PRC1) is an essential chromatin-modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small-molecule inhibitors with well-defined mechanisms of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary acute myeloid leukemia (AML) samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity.

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.

EWS-FLI1 and Menin Converge to Regulate ATF4 Activity in Ewing Sarcoma.

Ewing sarcomas are driven by EWS-ETS fusions, most commonly EWS-FLI1, which promotes widespread metabolic reprogramming, including activation of serine biosynthesis. We previously reported that serine biosynthesis is also activated in Ewing sarcoma by the scaffolding protein menin through as yet undefined mechanisms. Here, we investigated whether EWS-FLI1 and/or menin orchestrate serine biosynthesis via modulation of ATF4, a stress-response gene that acts as a master transcriptional regulator of serine biosynthesis in other tumors. Our results show that in Ewing sarcoma, ATF4 levels are high and that ATF4 modulates transcription of core serine synthesis pathway (SSP) genes. Inhibition of either EWS-FLI1 or menin leads to loss of ATF4, and this is associated with diminished expression of SSP transcripts and proteins. We identified and validated an EWS-FLI1 binding site at the ATF4 promoter, indicating that the fusion can directly activate ATF4 transcription. In contrast, our results suggest that menin-dependent regulation of ATF4 is mediated by transcriptional and post-transcriptional mechanisms. Importantly, our data also reveal that the downregulation of SSP genes that occurs in the context of EWS-FLI1 or menin loss is indicative of broader inhibition of ATF4-dependent transcription. Moreover, we find that menin inhibition similarly leads to loss of ATF4 and the ATF4-dependent transcriptional signature in MLL-rearranged B-cell acute lymphoblastic leukemia, extending our findings to another cancer in which menin serves an oncogenic role. IMPLICATIONS: These studies provide new insights into metabolic reprogramming in Ewing sarcoma and also uncover a previously undescribed role for menin in the regulation of ATF4.

Covalent inhibition of NSD1 histone methyltransferase.

The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

Covalent and noncovalent constraints yield a figure eight-like conformation of a peptide inhibiting the menin-MLL interaction.

The interaction between menin and mixed lineage leukemia (MLL) was identified as an interesting target for treating some cancers including acute leukemia. On the basis of the known crystal structure of the MBM1-menin complex (MBM - menin binding motif), several cyclic peptides were designed. Elaboration of the effective cyclization strategy using a metathesis reaction allowed for a successfully large number of derivatives to be obtained. Subsequent optimization of the loop size, as well as N-terminal, central and C-terminal parts of the studied peptides resulted in structures exhibiting low nanomolar activities. A crystal structure of an inhibitor-menin complex revealed a compact conformation of the ligand molecule, which is stabilized not only by the introduction of a covalent linker but also three intramolecular hydrogen bonds. The inhibitor adopts a figure eight-like conformation, which perfectly fits the cleft of menin. We demonstrated that the development of compact, miniprotein-like structures is a highly effective approach for inhibition of protein-protein interactions.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

Complexity of Blocking Bivalent Protein-Protein Interactions: Development of a Highly Potent Inhibitor of the Menin-Mixed-Lineage Leukemia Interaction.

The protein-protein interaction between menin and mixed-lineage leukemia 1 (MLL1) plays an important role in development of acute leukemia with translocations of the MLL1 gene and in solid tumors. Here, we report the development of a new generation of menin-MLL1 inhibitors identified by structure-based optimization of the thienopyrimidine class of compounds. This work resulted in compound 28 (MI-1481), which showed very potent inhibition of the menin-MLL1 interaction (IC50 = 3.6 nM), representing the most potent reversible menin-MLL1 inhibitor reported to date. The crystal structure of the menin-28 complex revealed a hydrogen bond with Glu366 and hydrophobic interactions, which contributed to strong inhibitory activity of 28. Compound 28 also demonstrates pronounced activity in MLL leukemia cells and in vivo in MLL leukemia models. Thus, 28 is a valuable menin-MLL1 inhibitor that can be used for potential therapeutic applications and in further studies regarding the role of menin in cancer.

Smad3-dependent CCN2 mediates fibronectin expression in human skin dermal fibroblasts.

The potential involvement of connective tissue growth factor (CCN2/CTGF) in extracellular matrix (ECM) production is recognized. However, the role CCN2 in fibronectin (FN) gene expression has remained incompletely understood and even controversial. Here we report that CCN2 is absolutely necessary for FN expression in primary human skin dermal fibroblasts, the major cells responsible for ECM production in skin. Gain- and loss-of-function approaches demonstrate that CCN2 is an essential component of FN expression in both basal and stimulation by TGF-ß signaling, the major regulator of FN expression. CCN2 is significantly induced by Smad3, a critical mediator of TGF-ß signaling. CCN2 acts as a downstream mediator of TGF-ß/Smad signaling and acting synergistically with TGF-ß to regulate FN gene expression. Finally, we observed that CCN2 and FN predominantly expressed in the dermis of normal human skin, stromal tissues of skin squamous cell carcinoma (SCC), and simultaneously induced in wounded human skin in vivo. These findings provide evidence that CCN2 is responsible for mediating the stimulatory effects of TGF-ß/Smad on FN gene expression, and attenuation of CCN2 expression may benefit to reduce fibrotic ECM microenvironment in disease skin.

Theoretical models of inhibitory activity for inhibitors of protein-protein interactions: targeting menin-mixed lineage leukemia with small molecules.

Development and binding affinity predictions of inhibitors targeting protein-protein interactions (PPI) still represent a major challenge in drug discovery efforts. This work reports application of a predictive non-empirical model of inhibitory activity for PPI inhibitors, exemplified here for small molecules targeting the menin-mixed lineage leukemia (MLL) interaction. Systematic ab initio analysis of menin-inhibitor complexes was performed, revealing the physical nature of these interactions. Notably, the non-empirical protein-ligand interaction energy comprising electrostatic multipole and approximate dispersion terms (E(10)El,MTP + EDas) produced a remarkable correlation with experimentally measured inhibitory activities and enabled accurate activity prediction for new menin-MLL inhibitors. Importantly, this relatively simple and computationally affordable non-empirical interaction energy model outperformed binding affinity predictions derived from commonly used empirical scoring functions. This study demonstrates high relevance of the non-empirical model we developed for binding affinity prediction of inhibitors targeting protein-protein interactions that are difficult to predict using empirical scoring functions.

Property Focused Structure-Based Optimization of Small Molecule Inhibitors of the Protein-Protein Interaction between Menin and Mixed Lineage Leukemia (MLL).

Development of potent small molecule inhibitors of protein-protein interactions with optimized druglike properties represents a challenging task in lead optimization process. Here, we report synthesis and structure-based optimization of new thienopyrimidine class of compounds, which block the protein-protein interaction between menin and MLL fusion proteins that plays an important role in acute leukemias with MLL translocations. We performed simultaneous optimization of both activity and druglike properties through systematic exploration of substituents introduced to the indole ring of lead compound 1 (MI-136) to identify compounds suitable for in vivo studies in mice. This work resulted in the identification of compound 27 (MI-538), which showed significantly increased activity, selectivity, polarity, and pharmacokinetic profile over 1 and demonstrated a pronounced effect in a mouse model of MLL leukemia. This study, which reports detailed structure-activity and structure-property relationships for the menin-MLL inhibitors, demonstrates challenges in optimizing inhibitors of protein-protein interactions for potential therapeutic applications.

Rational Design of Orthogonal Multipolar Interactions with Fluorine in Protein-Ligand Complexes.

Multipolar interactions involving fluorine and the protein backbone have been frequently observed in protein-ligand complexes. Such fluorine-backbone interactions may substantially contribute to the high affinity of small molecule inhibitors. Here we found that introduction of trifluoromethyl groups into two different sites in the thienopyrimidine class of menin-MLL inhibitors considerably improved their inhibitory activity. In both cases, trifluoromethyl groups are engaged in short interactions with the backbone of menin. In order to understand the effect of fluorine, we synthesized a series of analogues by systematically changing the number of fluorine atoms, and we determined high-resolution crystal structures of the complexes with menin. We found that introduction of fluorine at favorable geometry for interactions with backbone carbonyls may improve the activity of menin-MLL inhibitors as much as 5- to 10-fold. In order to facilitate the design of multipolar fluorine-backbone interactions in protein-ligand complexes, we developed a computational algorithm named FMAP, which calculates fluorophilic sites in proximity to the protein backbone. We demonstrated that FMAP could be used to rationalize improvement in the activity of known protein inhibitors upon introduction of fluorine. Furthermore, FMAP may also represent a valuable tool for designing new fluorine substitutions and support ligand optimization in drug discovery projects. Analysis of the menin-MLL inhibitor complexes revealed that the backbone in secondary structures is particularly accessible to the interactions with fluorine. Considering that secondary structure elements are frequently exposed at protein interfaces, we postulate that multipolar fluorine-backbone interactions may represent a particularly attractive approach to improve inhibitors of protein-protein interactions.

Pharmacologic inhibition of the Menin-MLL interaction blocks progression of MLL leukemia in vivo.

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.

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.

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.

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.

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.

Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin.

Aged human skin is fragile because of fragmentation and loss of type I collagen fibrils, which confer strength and resiliency. We report here that dermal fibroblasts express increased levels of collagen-degrading matrix metalloproteinases-1 (MMP-1) in aged (>80 years old) compared with young (21 to 30 years old) human skin in vivo. Transcription factor AP-1 and alpha2beta1 integrin, which are key regulators of MMP-1 expression, are also elevated in fibroblasts in aged human skin in vivo. MMP-1 treatment of young skin in organ culture causes fragmentation of collagen fibrils and reduces fibroblast stretch, consistent with reduced mechanical tension, as observed in aged human skin. Limited fragmentation of three-dimensional collagen lattices with exogenous MMP-1 also reduces fibroblast stretch and mechanical tension. Furthermore, fibroblasts cultured in fragmented collagen lattices express elevated levels of MMP-1, AP-1, and alpha2beta1 integrin. Importantly, culture in fragmented collagen raises intracellular oxidant levels and treatment with antioxidant MitoQ(10) significantly reduces MMP-1 expression. These data identify positive feedback regulation that couples age-dependent MMP-1-catalyzed collagen fragmentation and oxidative stress. We propose that this self perpetuating cycle promotes human skin aging. These data extend the current understanding of the oxidative theory of aging beyond a cellular-centric view to include extracellular matrix and the critical role that connective tissue microenvironment plays in the biology of aging.