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The repair of large-volume bone defects (LVBDs) remains a great challenge in the fields of orthopedics and maxillofacial surgery. Most clinically available bone-defect-filling materials lack proper degradability and efficient osteoinductivity. In this study, we synthesized a novel biomimetically-precipitated nanocrystalline calcium phosphate (BpNcCaP) with internally incorporated bone morphogenetic protein-2 (BpNcCaP + BMP-2) with an aim to develop properly degradable and highly osteoinductive granules to repair LVBDs. We first characterized the physicochemical properties of the granules with different incorporation amounts of BMP-2 using scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. We evaluated the cytotoxicity and cytocompatibility of BpNcCaP by assessing the viability and adhesion of MC3T3-E1 pre-osteoblasts using PrestoBlue assay, Rhodamine-Phalloidin and DAPI staining, respectively. We further assessed the

Large‐volume bone defects can result from congenital malformation, trauma, infection, inflammation and cancer. At present, it remains challenging to treat these bone defects with clinically available interventions. Allografts, xenografts and most synthetic materials have no intrinsic osteoinductivity, and so an alternative approach is to functionalize the biomaterial with osteoinductive agents, such as bone morphogenetic protein 2 (BMP2). Because it has been previously demonstrated that human salivary histatin‐1 (Hst1) promotes endothelial cell adhesion, migration and angiogenesis, we examine here whether Hst1 can promote BMP2‐induced bone regeneration. Rats were given subcutaneous implants of absorbable collagen sponge membranes seeded with 0, 50, 200 or 500 μg Hst1 per sample and 0 or 2 μg BMP2 per sample. At 18 days postsurgery, rats were sacrificed, and implanted regional tissue was removed for micro computed tomography (microCT) analyses of new bone (bone volume, trabecular number and trabecular separation). Four samples per group were decalcified and subjected to immunohistochemical staining to analyze osteogenic and angiogenic markers. We observed that Hst1 increased BMP2‐induced new bone formation in a dose‐dependent manner. Co‐administration of 500 μg Hst1 and BMP2 resulted in the highest observed bone volume and trabecular number, the lowest trabecular separation and the highest expression of osteogenic markers and angiogenic markers. Our results suggest that coadministration of Hst1 may enhance BMP2‐induced osteogenesis and angiogenesis, and thus may have potential for development into a treatment for large‐volume bone defects., Sufficient bone tissue is important for aesthetics and musculoskeletal functions. We showed that coadministration of human salivary histatin‐1 enhanced new bone formation (shown by microCT analysis) and angiogenesis (indicated by immunostaining of CD105, as shown by the black arrows) induced by bone morphogenetic protein 2 in a classic ectopic bone induction model using absorbable collagen sponge.

This paper uses analytic eclecticism to answer why the fragile status quo in the Taiwan Strait remains resilient. After pragmatically framing the research question, this research chooses three symbolic scenes in the Taiwan Strait: (1) Beijing’s economic statecraft towards Taiwan; (2) Taiwan’s soft power in finding the leeway out of its international dilemma; (3) The Sino-America competition over the narration in the cross-Strait relations. As a practice of eclecticism where the analytical framework is constructed based on each major school of international relations theories, this paper combines cases and theoretical tools to draw the whole picture of Taiwan issues. Ultimately, this research reached empirical and theoretical conclusions, calling for an innovative international relations theory that observes political phenomena conceptually and practically.

Development of adventitious roots (ARs) at the base of the shoot is an important adaptation of plants to waterlogging stress; however, its physiological mechanisms remain unclear. Here, we investigated the regulation of AR formation under waterlogged conditions by hormones and reactive oxygen species (ROS) in Cucumis sativus L., an agriculturally and economically important crop in China. We found that ethylene, auxin, and ROS accumulated in the waterlogged cucumber plants. On the other hand, application of the ethylene receptor inhibitor 1-methylcyclopropene (1-MCP), the auxin transport inhibitor 1-naphthylphthalamic acid (NPA), or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) decreased the number of ARs induced by waterlogging. Auxin enhanced the expression of ethylene biosynthesis genes, which led to ethylene entrapment in waterlogged plants. Both ethylene and auxin induced the generation of ROS. Auxin-induced AR formation was inhibited by 1-MCP, although ethylene-induced AR formation was not inhibited by NPA. Both ethylene- and auxin-induced AR formation were counteracted by DPI. These results indicate that auxin-induced AR formation is dependent on ethylene, whereas ethylene-induced AR formation is independent of auxin. They also show that ROS signals mediate both ethylene- and auxin-induced AR formation in cucumber plants.

Simple Summary MLL-rearranged leukemia, driven by MLL-fusion proteins, is an aggressive, therapy-resistant leukemia found in >60% of infant leukemia and ~10% of adult leukemia. Studies have shown that inhibiting DOT1L enzymatic activity blocks leukemogenesis. However, DOT1L is critical for various normal cellular functions, including hematopoiesis. This study aimed to show that targeting the interaction between the MLL-AF9 fusion and DOT1L would inhibit leukemogenesis while sparing non-leukemic hematopoiesis. We found that disrupting the AF9-DOT1L interaction with a single point mutation was sufficient to impair leukemogenesis. We also demonstrate that genetic interventions that result in loss of DOT1L enzymatic activity in non-leukemic cells rapidly depletes hematopoietic stem and progenitor cells within 7–10 days; however, hematopoiesis was preserved when the AF9-DOT1L interaction was disrupted, leaving the enzymatic function intact. These studies are a proof of concept demonstrating the potential therapeutic advantage of inhibiting the AF9-DOT1L interaction and disrupting the integrity of the MLL-fusion complex. Abstract MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9–DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

To determine the electroclinical features of fixation-off sensitivity (FOS) in patients with idiopathic generalized epilepsy (IGE). We searched the EEG database using the terms "fixation-off sensitivity" and "idiopathic generalized epilepsy" over a four-year period from March 2014 to April 2018 in the Xijing Hospital, Xi'an, China. FOS was evaluated according to the technique proposed by Panayiotopoulos. Photic stimulation procedure and neuropsychological testing were performed during video-EEG monitoring. FOS was observed in eight patients with several different IGE syndromes, including four with eyelid myoclonia/Jeavons syndrome, two with juvenile myoclonic epilepsy, one with photosensitivity epilepsy, and one with epilepsy with generalized tonic-clonic seizures only. FOS was associated with seizures in five patients manifesting with eyelid myoclonic, myoclonic, and myoclonic-tonic-clonic seizures, and eyelid myoclonic status. FOS coexisted with photosensitivity in six patients as independent EEG features. Neuropsychological testing revealed transitory cognitive impairments associated with FOS. FOS is associated with several different IGE syndromes and may coexist with photosensitivity in the same patient as independent EEG features. FOS may be associated with both clinical seizures and cognitive impairments. Intermittent photic stimulation and registration of different eye conditions with and without fixation will aid the study of the dynamics of the visual system in epilepsy patients. [Published with video sequences on www.epilepticdisorders.com].

Interactions between protein ligands and receptors play crucial roles in cell-cell signalling. Most of the human cell surface receptors have been identified in the post-Human Genome Project era but many of their corresponding ligands remain unknown. To facilitate the pairing of orphan receptors, 2762 sequences encoding all human single-pass transmembrane proteins were selected for inclusion into a mammalian-cell expression library. This expression library, consisting of all the individual extracellular domains (ECDs), was constructed as a Fab fusion for each protein. In this format, individual ECD can be produced as a soluble protein or displayed on cell surface, depending on the applied heavy-chain Fab configuration. The unique design of the Fab fusion concept used in the library led to not only superior success rate of protein production, but also versatile applications in various high-throughput screening paradigms including protein-protein binding assays as well as cell binding assays, which were not possible for any other existing expression libraries. The protein library was screened against human coagulation factor VIIa (FVIIa), an approved therapeutic for the treatment of hemophilia, for binding partners by AlphaScreen and ForteBio assays. Two previously known physiological ligands of FVIIa, tissue factor (TF) and endothelial protein C receptor (EPCR) were identified by both assays. The cell surface displayed library was screened against V-domain Ig suppressor of T-cell activation (VISTA), an important immune-checkpoint regulator. Immunoglobulin superfamily member 11 (IgSF11), a potential target for cancer immunotherapy, was identified as a new and previously undescribed binding partner for VISTA. The specificity of the binding was confirmed and validated by both fluorescence-activated cell sorting (FACS) and surface plasmon resonance (SPR) assays in different experimental setups.

Introduction This paper set out to demonstrate the clinical features of IGE patients coexisting with FOS, meanwhile to evaluate the consciousness and higher cognitive functions during the long term generalized epileptic discharges elicited by elimination of central vision and fixation. Methods The database of EEG records was searched for FOS and IGE over 4 years from January 2014 to November 2017 at Xijing hospital, Xi’an, China. FOS was evaluated according to the technique proposed by Panayiotopoulos. Photic stimulation procedure was performed according the methodology of updated European algorithm for visual stimulation. In order to evaluate the degree of possible transient impaired consciousness and cognitive function caused by the epileptic discharges, neuropsychological evaluation was performed during video-EEG monitoring with eyes open and with eye closed according the procedure developed by the European taskforce. For comparison, performance during eye open period was set as the normal baseline. Results We identified five (four males and one female) young IGE patients with FOS (one GTCS-only; one JME, two EMA/JS, one IGPE photosensitivity epilepsy). Three of them had family history of epilepsy. All the patients presented with FOS, two of them manifest with FOS associated with seizures captured by video-EEG, four of them coexisted with photosensitivity (two confirmed, two probable). The GTCS was well managed on AED. Two patients complained the GTCS were only provoked by and emotional stress, one patient complained seizures were only triggered by television, the other two patients reported no precipitating factors. The EEG features of FOS presented as high amplitude generalized epileptic discharge predominantly over frontal and occipital regions. Consciousness (awareness and responsiveness) looked like well-preserved during longstanding generalized epileptic discharge induced by elimination of visual fixation, while mild transitory impairment of higher cognitive functions was observed when neuropsychological testing was performed. Conclusion (1) The fixation-off phenomenon as a reflex EEG phenomenon in IGE may either manifest with seizures or represent an EEG abnormality associated with higher cortical cognitive impairment. (2) Neuropsychological assessment can make a highly contribution to the understanding of the mechanisms of consciousness and higher cortical cognitive function in epilepsy. Both concomitant Video-EEG and high-precision neuropsychological testing are necessary to detect the subtle symptom. (3) FOS may be not self-limiting, could occur in several different idiopathic generalized epilepsy syndromes, and therefore, their occurrence alone is not sufficient to characterize a definite epilepsy syndrome.

The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865–874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia. Background: MLL fusion proteins use similar strategy for leukemic transformation through DOT1L recruitment. Results: Ten amino acids in DOT1L were identified as essential for binding and transformation by MLL-AF9. Conclusion: Biochemical and functional results indicate that blocking DOT1L recruitment represents a promising therapeutic strategy for mixed lineage leukemia. Significance: Identified DOT1L peptide will lay a foundation toward discovery of chemical tools able to block DOT1L recruitment.

Targeting the baculoviral inhibitor of apoptosis proteins repeat (BIR) 3 of X-linked inhibitor of apoptosis proteins (XIAP) represents an innovative strategy for the design of chemosensitizers. Acylated flavonol monorhamnosides (AFMR) from Eriobotrya japonica Lindl. (Rosaceae) were virtually predicted as ligands of the XIAP BIR3 domain by using a previously generated pharmacophore model. From the methanol leaf extract of E. japonica an enriched mixture of AFMR was obtained showing chemosensitizing potential in combination with etoposide in XIAP-overexpressing Jurkat cells. The HPLC-SPE-NMR hyphenated technique facilitated the structure elucidation of three known and two new natural AFMR. The main constituent and virtual hit, kaempferol-3-O-α-l-(2″,4″-di-E-p-coumaroyl)-rhamnoside (3) was isolated from the enriched fraction. Applying a fluorescence polarization based binding assay, 3 was identified as XIAP BIR3 ligand with a dose-dependent affinity (IC₅₀ 10.4 μM). Further, 3 induced apoptosis in XIAP-overexpressing Jurkat cells and activated caspase-9 in combination with etoposide. Docking experiments revealed a major impact of the coumaric acid and sugar moieties of 3 on XIAP BIR3 binding, which was experimentally confirmed. To conclude, this study elucidates 3 as natural, small-molecular weight XIAP BIR3 inhibitor using a combination of in silico and HPLC-SPE-NMR hyphenated techniques.

The effects of La3+ on the unfolded protein response signaling pathways were investigated in human hepatoblastoma HepG2 cells. Our data showed that La3+ could induce unfolded protein response in HepG2 cells, including a significant increase of BiP/GRP78 level, which is an important ER residential chaperone and an ER stress hallmark, in a concentration and time-dependent manner, UPR transducer IRE1 phosphorylation and splicing activation IRE1 downstream substrate XBP1 mRNA. By using La3+-affinity chromatography, the possible cellular target of La3+ leading to UPR events was shown to be the ER residential chaperone BiP/GRP78. BiP/GRP78 was shown to be a La3+ binding protein and the interaction of La3+ with BiP/GRP78 resulted in dissociation of BiP-IRE1 complexes. La3+ induced dissociation of the BiP/GRP78-IRE1 complex was in a time and concentration manner. The apparent dissociation constant was estimated to be 4 nM. In addition, La3+ was observed to slightly stimulate the production of cellular ROS and cause alteration of intracellular Ca2+, indicating the possible involvement of ROS and Ca2+ alteration in La3+ induced UPR. The present work provides a new perspective for understanding the biological and toxicological effects of La3+.

Mixed lineage leukemia (MLL) is an aggressive form of leukemia where the MLL gene is translocated and fused to more than 80 different nuclear, cytoplasmic and membrane proteins. MLL-AF9 is one of the most common MLL-fusions. This fusion partner is known to recruit several multiprotein complexes including the AEP, SEC and DOT1L complexes leading to transcriptional activation. In particular, the recruitment of Disruptor of telomeric silencing 1-like (DOT1L), a H3K79 histone methyltransferase, is essential for leukemogenesis by multiple MLL fusion proteins. We mapped the binding site to a 10 amino acid segment (865 - 874) on DOT1L where AF9 binds. Peptides derived from this region that show dose-dependent disruption of this interaction. We also demonstrated that DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. Encouraged by these results, we generated both genetic and chemical tools to elucidate the role of DOT1L recruitment to the MLL fusion partners and the mechanism of leukemogenic inhibition by disrupting the protein-protein interaction (PPI) between MLL-AF9 and DOT1L. Fl/Fl DOT1L MLL-AF9 Cre+ cell lines were generated with different constructs of DOT1L. These constructs consisted of DOT1L mutants lacking the 10 amino acid binding site, a I867A point mutant known to block DOT1L binding, and an enzymatic mutant known to yield a catalytically inactive protein. As control cell lines, MLL-AF6, a MLL-fusion containing a cytoplasmic protein, and E2A-HLF, a non-DOT1L dependent fusion, were generated to demonstrate the specific effects of generated DOT1L mutant constructs. Both DOT1L PPI mutants impaired the transformation by MLL-AF9 and induced cell death by inducing apoptosis and cell cycle arrest similarly to enzymatic inhibition. These results established a foundation for discovering small-molecule inhibitors that disrupting the AF9-DOT1L as potential disease-specific therapies that target chromatin modifications in this highly aggressive leukemia. A high throughput screening was conducted identifying several different chemical classes of small molecules that bind to the AF9 C-terminal hydrophobic binding site and disrupt the PPI between DOT1L and MLL-AF9 fusion protein. Identified small molecule inhibitors were validated with series of biochemical, functional and cell-based assays. Validated compounds selectively inhibit the growth of the DOT1L dependent murine cells and induce cell death in a similar manner to the genetic approach. The small molecules also showed specificity in killing human MLL-fusion cell lines in comparison to non-MLL fusion leukemia. These results show that blocking the recruitment of DOT1L by AF9 using both genetic and chemical tools eliminate MLL-AF9 mediated immortalization emphasizing an essential function for this interaction in leukemogenesis and warrant further development of the identified small-molecule inhibitors. Citation Format: Sierrah M. Grigsby, James Ropa, Justin Serio, Chenxi Shen, Jennifer Chase, Ivan Maillard, Andrew Muntean, Zaneta Nikolovska-Coleska. Using genetic and chemical approaches to probe the mechanism of DOT1L recruitment in MLL fusion leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2017-5489

Mcl-1, an antiapoptotic member of the Bcl-2 family of proteins, is a validated and attractive target for cancer therapy. Overexpression of Mcl-1 in many cancers results in disease progression and resistance to current chemotherapeutics. Utilizing high-throughput screening, compound 1 was identified as a selective Mcl-1 inhibitor and its binding to the BH3 binding groove of Mcl-1 was confirmed by several different, but complementary, biochemical and biophysical assays. Guided by structure-based drug design and supported by NMR experiments, comprehensive SAR studies were undertaken and a potent and selective inhibitor, compound 21, was designed which binds to Mcl-1 with a Ki of 180 nM. Biological characterization of 21 showed that it disrupts the interaction of endogenous Mcl-1 and biotinylated Noxa-BH3 peptide, causes cell death through a Bak/Bax-dependent mechanism, and selectively sensitizes Eμ-myc lymphomas overexpressing Mcl-1, but not Eμ-myc lymphoma cells overexpressing Bcl-2. Treatment of human leukemic cell lines with compound 21 resulted in cell death through activation of caspase-3 and induction of apoptosis.

Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangements of the MLL1 gene generate fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. To gain insights into the unique functions of DOT1L in MLL-driven leukemia, we elucidated the mechanisms of DOT1L recruitment to the MLL fusion partners. The binding site was mapped to a short segment of 10 amino acids in DOT1L and peptides derived from this region disrupted the interaction between DOT1L and MLL-AF9. DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. This discovery has established a foundation for disease-specific therapies that target chromatin modifications in highly malignant leukemias. Applying high throughput screening approach several different chemical classes of small molecules that disrupt the protein-protein interactions between DOT1L and oncogenic MLL-fusion proteins were identified and validated. To evaluate if the AF9-binding domain of DOT1L is critical for its functions in normal hematopoietic stem cells as opposed to leukemias driven by MLL fusion proteins, genetic tools were developed to functionally investigate the importance of the DOT1L AF9-binding domain in MLL-AF9-driven leukemia and its role in the physiological functions of DOT1L in normal hematopoiesis. Our findings demonstrate that pharmacological inhibition of the DOT1L complex through disrupting the AF9-DOT1L interactions may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression. Citation Format: Sierrah Grigsby, Jennifer Chase, James Ropa, Justin Serio, Chenxi Shen, Martha Larsen, Preston Donover, Melvin Reichman, Andrew Muntean, Ivan Maillard, Zaneta Nikolovska-Coleska. Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3803.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Myeloid cell leukemia-1 (Mcl-1) is a potent anti-apoptotic protein, a member of the prosurvival Bcl-2 family, and its role is emerging as a critical survival factor in a broad range of human cancers. Mcl-1 represents a very attractive molecular target for development of a new class of cancer therapy and there is still need for developing BH3 mimetics that can efficiently and selectively target Mcl-1 protein. We employed integrated screening approach through combining high throughput and virtual screening, to identify novel chemical classes as Mcl-1 inhibitors. For this purpose we developed a dual-readout HTS assay that combines two assay technologies, FP and FRET, into one system using the Mcl-1 and labeled Noxa BH3 derived peptide and optimized in a 1,536-well ultra-HTS format. This assay was used for screening a library of 102,255 compounds. As two assay platforms were utilized for the same target simultaneously, hit information was enriched identifying 1214 compounds. To further improve the output and the quality of the identified inhibitors, as well as to incorporate the structure-based knowledge of the interactions between Mcl-1 and number of BH3 peptides, we have integrated in silico target-based screening for selection of the most promising hits for further validation. The complex structure between Mcl-1 and Noxa BH3 peptide (PDB ID: [2NLA][1]) was used for the induced fit docking of 1214 hits. Followed by the scoring and ranking of the identified hits, 62 compounds were selected for further evaluation in a series of complementary biochemical, biophysical and functional assays. Several in vitro binding assays with different platform, FP, SPR, and (HSQC) NMR spectroscopy, were used to determine the binding affinity of the compounds. The binding data revealed that we have identified inhibitors with different selectivity profiles against five members of the Bcl-2 family: Mcl-1, Bcl-2, Bcl-xL, Bcl-w and A1. 15N HSQC spectra conclusively showed that newly identified compounds interact with the BH3 domain in Mcl-1 protein and affect many residues in the BH3 binding groove. Using pull down assay it was demonstrated that the identified compounds were able in a dose dependent manner to disrupt interactions between endogenous Mcl-1 protein and biotin labeled Noxa BH3 peptide. Functional studies showed that compounds can antagonize Mcl-1 function and induce cytochrome c and Smac release from the isolated mitochondria. By using murine embryonic fibroblasts (MEFs), wild type and deficient in both Bax and Bak (double knock out), it was further demonstrated that the cytotoxic activity and induction of apoptosis, depend on Bax and/or Bak, suggesting that the compounds function as BH3 mimetics. Collectively, these findings provide several different chemical classes for further chemical modifications and optimization toward developing a new class of anticancer drugs as Mcl-1 inhibitors. Citation Format: Ahmed Mady, Chenzhong Liao, Fardokht Abulwerdi, Chenxi Shen, Yuhong Du, Jeanne Stuckey, Haian Fu, Zaneta Nikolovska-Coleska. Identification of novel Mcl-1 inhibitors using integrated screening approach: combining high throughput and virtual screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2013-2469 [1]: /lookup/external-ref?link_type=PDB&access_num=2NLA&atom=%2Fcanres%2F73%2F8_Supplement%2F2469.atom

MLL fusion proteins activate target genes in part via recruitment of the histone-modifying enzyme, DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase with selective activity for histone H3 lysine 79 (H3K79). The resulting hypermethylation of H3K79 at the HOX and MEIS1 loci appears to be pivotal for leukemogenesis in both AML and ALL. These findings emphasize the central role of DOT1L in leukemogenesis and strongly suggest that pharmacologic inhibition of DOT1L could be of therapeutic benefit for a number of ALL and AML subtypes. Applying a biochemical screening approach, two classes of small molecule DOT1L substrate competitive inhibitors were discovered. They selectively inhibit the cell growth in leukemic cells with MLL-translocation, in comparison with leukemic cells without MLL translocation, resulting in a dose and time dependent decrease of cellular H3K79 methylation, decrease in expression of MLL-target genes HOXA9 and MEIS1, inducing G0/G1 cell cycle arrest, an increase in expression of differentiation markers and death by apoptosis. Although the DOT1L inhibitors showed no toxicity on normal bone marrow, knockout studies show that DOT1L deficiency is associated with chromosomal instability and fatal progressive anemia. This suggested that developing therapeutic strategies other than global inhibition of DOT1L activity may be necessary. Therefore we characterized the protein-protein interaction (PPI) between DOT1L and ENL/AF9 and assessed its importance on MLL fusion protein-mediated transformation. We found that ENL and AF9 C-terminal domains bind to DOT1L with Kd of 238 nM and 111 nM respectively. We have mapped the binding site in DOT1L and found that only 10 amino acid of DOT1L are involved in interaction with ENL and AF9. Synthetic DOT1L 10mer peptide binds ENL/AF9 and disrupts the interaction between DOT1L/ENL and DOT1L/AF9. Alanine scanning mutagenesis studies showed that four hydrophobic residues are detrimental for the binding of DOT1L to both proteins ENL/AF9. To examine the critical role of the DOT1L-AF9 interaction in transformation by MLL fusion protein, colony forming unit (CFU) assay was performed using MLL-AF9 transformed cells lacking endogenous DOT1L. The colony forming potential of the MLL-immortalized cells was completely abolished by introducing DOT1L construct lacking 10 amino acid AF9 interacting residues (DOT1L (Δ10aa)), while the wild type DOT1L construct was able to restore CFU formation. Importantly global levels of H3K79 methylation were equivalent, but only the DOT1L (Δ10aa) construct failed to restore CFU formation. These results demonstrate that DOT1L interaction with MLL-AF9 in addition to H3K79 methylation is required for transformation of MLL-AF9. This suggests that selective disruption of this PPI is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL-fusion protein associated leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A158.

Myeloid cell leukemia (Mcl-1) is a multidomain, anti-apoptotic protein, member of the Bcl-2 family proteins, an important survival factor for many cancers. Mcl-1 has a critical and distinct role(s) in maintaining cell survival and emerging as an independent and promising therapeutic target. Through high throughout screening (HTS) of 53,300 structurally diverse synthetic organic compounds, we have discovered a number of novel inhibitors of Mcl-1. The hits identified by HTS were evaluated in a series of complementary biochemical, biophysical, functional and cellular assays to eliminate false positives and to determine the specificity and mechanism of action of these new compounds and 23 compounds were found to be inhibitors of Mcl-1. The most promising compounds, 59 and 62, have diverse chemical scaffolds. We have shown that these small molecule inhibitors bind to the BH3 binding site in Mcl-1 with IC50 values of 1,200 nM and 200 nM, respectively, and compete with BH3 peptides derived from Bid, Bim, or Noxa proteins. They bind to the Mcl-1 protein showing selectivity over two other Bcl-2 family members, Bcl-2 and Bcl-xL. NMR spectroscopy shows that 59 and 62 bind to the same BH3 domain of Mcl-1 as the Bim BH3 peptide. These initial lead compounds antagonize Mcl-1 on the functional level and they induce release of cytochrome c, inhibit cell growth and induce apoptosis in pancreatic and melanoma cancer cells with high levels of Mcl-1. We have tested several analogues of 59 and 62, both synthetic and commercially available, and established initial structure-activity relationships. One of the compounds tested (77), an analogue of 59, has improved binding affinity to Mcl-1 protein, as well as potent activity in inhibition of cell growth in cancer cells. Furthermore, by using murine embryonic fibroblasts (MEFs), wild type and deficient in both Bax and Bak (double knock out), it was demonstrated that the cytotoxic activity and induction of apoptosis by several analogues of the lead compound 59, depend on Bax and/or Bak, suggesting that they function as BH3 mimetics. Collectively, these findings provide good promise for chemical modifications of the new identified lead compounds, 59 and 62, and their further optimization toward developing a new class of anticancer drugs, Mcl-1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4521.