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.

Mutant NPM1 Hijacks Transcriptional Hubs to Maintain Pathogenic Gene Programs in Acute Myeloid Leukemia.

Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of biological functions. In 30% of acute myeloid leukemia (AML), the terminal exon of NPM1 is often found mutated, resulting in the addition of a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have aberrant expression of the HOXA/B genes, whose overexpression leads to leukemogenic transformation. Here, for the first time, we comprehensively prove that NPM1c binds to a subset of active gene promoters in NPM1c AMLs, including well-known leukemia-driving genes-HOXA/B cluster genes and MEIS1. NPM1c sustains the active transcription of key target genes by orchestrating a transcription hub and maintains the active chromatin landscape by inhibiting the activity of histone deacetylases. Together, these findings reveal the neomorphic function of NPM1c as a transcriptional amplifier for leukemic gene expression and open up new paradigms for therapeutic intervention. SIGNIFICANCE: NPM1 mutation is the most common mutation in AML, yet the mechanism of how the mutant protein results in AML remains unclear. Here, for the first time, we prove mutant NPM1 directly binds to active chromatin regions and hijacks the transcription of AML-driving genes. See related article by Uckelmann et al., p. 746. This article is highlighted in the In This Issue feature, p. 517.

Correction: Ecological restoration stimulates environmental outcomes but exacerbates water shortage in the Loess Plateau.

[This corrects the article DOI: 10.7717/peerj.13658.].

Ecological restoration stimulates environmental outcomes but exacerbates water shortage in the Loess Plateau.

Restoration is the natural and intervention-assisted set of processes designed to promote and facilitate the recovery of an ecosystem that has been degraded, damaged, or destroyed. However, it can also have an adverse effect on the environment. Thus, assessing an ecological restoration project's impact is crucial to determining its success and optimum management strategies. We performed a meta-analysis concerning the environmental outcomes during the years 2000-2015 resulting from the "Grain for Green" Project (GFGP) implementation in the Loess Plateau (LP). Data were gathered from 40 peer-reviewed English-language articles chosen from a pool of 332 articles. The results showed that, on average, GFGP increased forest coverage by 35.7% (95% CI [24.15-47.52%]), and grassland by 1.05% (95% CI [0.8-1.28%]). At the same time, GFGP has a positive impact on soil carbon (C) sequestration, net ecosystem production (NEP), and net primary production (NPP), from the years 2000 to 2015 by an average of 36% (95% CI [28.96-43.18%]), 22.7% (95% CI [9.10-36.79%]), and 13.5% (95% CI [9.44-17.354%]), respectively. Soil erosion, sediment load, runoff coefficient, and water yield were reduced by 13.3% (95% CI [0.27-25.76%]), 21.5% (95% CI [1.50-39.99%]), 22.4% (95% CI [5.28-40.45%]) and 43.3% (95% CI [27.03-82.86%]), respectively, from the years 2000 to 2015. Our results indicate that water supply decreased with the increase of vegetation coverage. Therefore, to balance the needs for green space, GFGP policies and strategies should recover, enhance, and sustain more resilient ecosystems.

Intensive vegetable production results in high nitrate accumulation in deep soil profiles in China.

A comprehensive understanding of the patterns and controlling factors of nitrate accumulation in intensive vegetable production is essential to solve this problem. For the first time, the national patterns and controlling factors of nitrate accumulation in soil of vegetable systems in China were analysed by compiling 1262 observations from 117 published articles. The results revealed that the nitrate accumulation at 0-100 cm, 100-200 cm, 200-300 cm, and >300 cm were 504, 390, 349, and 244 kg N ha-1, with accumulation rates of 62, 54, 19, and 16 kg N ha-1 yr-1 for plastic greenhouse vegetables (PG); for open field vegetables (OF), they were 264, 217, 228, and 242 kg N ha-1 with accumulation rates of 26, 24, 18, and 10 kg N ha-1 yr-1, respectively. Nitrate accumulation at 0-100 cm, 0-200 cm, and 0-400 cm accounted for 5%, 11%, and 17% of accumulated nitrogen (N) inputs for PG, and represented 4%, 9%, and 13% of accumulated N inputs for OF. Nitrogen input rates and soil pH had positive effects and soil organic carbon, water input rate, and carbon to nitrogen ratio (C/N) had negative effects on nitrate accumulation in root zone (0-100 cm soil). Nitrate accumulation in deep vadose zone (>100 cm soil) was positively correlated with N and water input rates, and was negatively correlated with soil organic carbon, C/N, and the clay content. Thus, for a given vegetable soil with relatively stable soil pH and soil clay content, reducing N and water inputs, and increasing soil organic carbon and C/N are effective measures to control nitrate accumulation.

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.

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.

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.

HOXA9 Reprograms the Enhancer Landscape to Promote Leukemogenesis.

Aberrant expression of HOXA9 is a prominent feature of acute leukemia driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid and B progenitor cells leads to significant enhancer reorganizations with prominent emergence of leukemia-specific de novo enhancers. Alterations in the enhancer landscape lead to activation of an ectopic embryonic gene program. We show that HOXA9 functions as a pioneer factor at de novo enhancers and recruits CEBPα and the MLL3/MLL4 complex. Genetic deletion of MLL3/MLL4 blocks histone H3K4 methylation at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis in vivo. These results suggest that therapeutic targeting of HOXA9-dependent enhancer reorganization can be an effective therapeutic strategy in acute leukemia with HOXA9 overexpression.

GAS41 Recognizes Diacetylated Histone H3 through a Bivalent Binding Mode.

GAS41 is a chromatin-associated protein that belongs to the YEATS family and is involved in the recognition of acetyl-lysine in histone proteins. A unique feature of GAS41 is the presence of a C-terminal coiled-coil domain, which is responsible for protein dimerization. Here, we characterized the specificity of the GAS41 YEATS domain and found that it preferentially binds to acetylated H3K18 and H3K27 peptides. Interestingly, we found that full-length, dimeric GAS41 binds to diacetylated H3 peptides with an enhanced affinity when compared to those for monoacetylated peptides, through a bivalent binding mode. We determined the crystal structure of the GAS41 YEATS domain with H3K23acK27ac to visualize the molecular basis of diacetylated histone binding. Our results suggest a unique binding mode in which full-length GAS41 is a reader of diacetylated histones.

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.

Pharmacologic Inhibition of the Menin-MLL Interaction Leads to Transcriptional Repression of PEG10 and Blocks Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for approximately 85% of malignant liver tumors and results in 600,000 deaths each year, emphasizing the need for new therapies. Upregulation of menin was reported in HCC patients and high levels of menin correlate with poor patient prognosis. The protein-protein interaction between menin and histone methyltransferase mixed lineage leukemia 1 (MLL1) plays an important role in the development of HCC, implying that pharmacologic inhibition of this interaction could lead to new therapeutic strategy for the HCC patients. Here, we demonstrate that the menin-MLL inhibitor MI-503 shows antitumor activity in in vitro and in vivo models of HCC and reveals the potential mechanism of menin contribution to HCC. Treatment with MI-503 selectively kills various HCC cell lines and this effect is significantly enhanced by a combination of MI-503 with sorafenib, the standard-of-care therapy for HCC. Furthermore, MI-503 reduces sphere formation and cell migration in in vitro HCC models. When applied in vivo, MI-503 gives a strong antitumor effect both as a single agent and in combination with sorafenib in mice xenograft models of HCC. Mechanistically, treatment with MI-503 downregulates expression of several genes known to play a critical role in proliferation and migration of HCC cells, including PEG10, and displaces the menin-MLL1 complex from the PEG10 promoter, resulting in reduced H3K4 methylation and transcriptional repression. Overall, our studies reveal a mechanistic link between menin and genes involved in HCC and demonstrate that pharmacologic inhibition of the menin-MLL interaction might represent a promising therapeutic approach for HCC. Mol Cancer Ther; 17(1); 26-38. ©2017 AACR.

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.

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.

C/EBPα is an essential collaborator in Hoxa9/Meis1-mediated leukemogenesis.

Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, almost always in association with overexpression of its cofactor meis homeobox 1 (MEIS1) . A wide range of data suggests that HOXA9 and MEIS1 play a synergistic causative role in AML, although the molecular mechanisms leading to transformation by HOXA9 and MEIS1 remain elusive. In this study, we identify CCAAT/enhancer binding protein alpha (C/EBPα) as a critical collaborator required for Hoxa9/Meis1-mediated leukemogenesis. We show that C/EBPα is required for the proliferation of Hoxa9/Meis1-transformed cells in culture and that loss of C/EBPα greatly improves survival in both primary and secondary murine models of Hoxa9/Meis1-induced leukemia. Over 50% of Hoxa9 genome-wide binding sites are cobound by C/EBPα, which coregulates a number of downstream target genes involved in the regulation of cell proliferation and differentiation. Finally, we show that Hoxa9 represses the locus of the cyclin-dependent kinase inhibitors Cdkn2a/b in concert with C/EBPα to overcome a block in G1 cell cycle progression. Together, our results suggest a previously unidentified role for C/EBPα in maintaining the proliferation required for Hoxa9/Meis1-mediated leukemogenesis.

Expression of the tissue inhibitor of metalloproteinase-3 by transplanted VSMCs modifies heart structure and function after myocardial infarction.

OBJECTIVES: Extracellular matrix (ECM) remodelling is a critical aspect of cardiac remodelling following myocardial infarction. Tissue inhibitors of metalloproteinases (TIMPs) are physiological inhibitors of matrix metalloproteinases (MMPs) that degrade the ECM proteins. TIMP-3 is highly expressed in the heart and is markedly downregulated in patients with ischaemic cardiomyopathy. Cell-based gene therapy can enhance the effects of cell transplantation by temporally and spatially regulating the release of the gene product. The purpose of this study was to investigate the role of TIMP-3 gene-transfected vascular smooth muscle cells (VSMCs) in modifying heart structure and function in rats when transplanted 3days after myocardial infarction (MI). METHODS: Anesthetised rats were subjected to coronary artery ligation followed 3days later by thoracotomy and transplantation of TIMP-3 gene-transfected VSMCs, untransfected VSMCs or medium injected directly into the ischaemic myocardium. We assessed left ventricular structure and function by echocardiography and morphometry, and measured the levels of myocardial matrix metalloproteinase-2 and -9 (MMP-2, MMP-9), TIMP-3 and tumour necrosis factor-α (TNF-α) at 4weeks post-myocardial infarction. RESULTS: Transplantation of TIMP-3 gene-transfected VSMCs and untransfected VSMCs significantly decreased scar expansion and ventricular dilatation 25days post-transplantation (4weeks after MI). MMPs and TNF-α levels were reduced in the transplantation groups when compared to the group that was given an injection of medium only. Transplantation of TIMP-3 gene-transfected VSMCs was more effective in preventing progressive cardiac dysfunction, ventricular dilatation and in reducing MMP-2, MMP-9 and TNF-α levels when compared to the transplantation of untransfected VSMCs. CONCLUSIONS: TIMP-3 gene transfection was associated with attenuated left ventricular dilation and recovery of systolic function after MI compared with the control. TIMP-3 transfection enhanced the effects of transplanted VSMCs in rats by inhibiting matrix degradation and inflammatory cytokine expression, leading to improved myocardial remodelling.

SRA gene knockout protects against diet-induced obesity and improves glucose tolerance.

We have recently shown that the non-coding RNA, steroid receptor RNA activator (SRA), functions as a transcriptional coactivator of PPARγ and promotes adipocyte differentiation in vitro. To assess SRA function in vivo, we have generated a whole mouse Sra1 gene knock-out (SRA(-/-)). Here, we show that the Sra1 gene is an important regulator of adipose tissue mass and function. SRA is expressed at a higher level in adipose tissue than other organs in wild type mice. SRA(-/-) mice are resistant to high fat diet-induced obesity, with decreased fat mass and increased lean content. This lean phenotype of SRA(-/-) mice is associated with decreased expression of a subset of adipocyte marker genes and reduced plasma TNFα levels. The SRA(-/-) mice are more insulin sensitive, as evidenced by reduced fasting insulin, and lower blood glucoses in response to IP glucose and insulin. In addition, the livers of SRA(-/-) mice have fewer lipid droplets after high fat diet feeding, and the expression of lipogenesis-associated genes is decreased. To our knowledge, these data are the first to indicate a functional role for SRA in adipose tissue biology and glucose homeostasis in vivo.