Tranylcypromine-Based LSD1 Inhibitors: Structure-Activity Relationships,Antiproliferative Effects in Leukemia,and Gene Target Modulation

Abstract : LSD1 is a lysine demethylase highly involved in initiation and development of cancer. To design highly effective covalent inhibitors, a strategy is to fill its large catalytic cleft by designing tranylcypromine (TCP) analogs decorated with long, hindered substituents. We prepared three series of TCP analogs, carrying aroyl- and arylacetylamino ( 1 a – h ), Z-amino acylamino ( 2 a – o ), or double-substituted benzamide ( 3 a – n ) residues at the C4 or C3 position of the phenyl ring. Further fragments obtained by chemical manipulation applied on the TCP scaffold (compounds 4 a – i ) were also prepared. When tested against LSD1, most of 1 and 3 exhibited IC₅₀ values in the low nanomolar range, with 1 e and 3 a , d , f , g being also the most selective respect to monoamine oxidases. In MV4-11 AML and NB4 APL cells compounds 3 were the most potent, displaying up to sub-micromolar cell growth inhibition against both cell lines ( 3 a ) or against NB4 cells ( 3 c ). The most potent compounds in cellular assays were also able to induce the expression of LSD1 target genes, such as GFI-1b, ITGAM, and KCTD12, as functional read-out for LSD1 inhibition. Mouse and human intrinsic clearance data highlighted the high metabolic stability of compounds 3 a , 3 d and 3 g . Further studies will be performed on the new compounds 3 a and 3 c to assess their anticancer potential in different cancer contexts.     

Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells

Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR⁺. The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2-nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches.     

Targeting Casein Kinase 1 (CK1) in Hematological Cancers

The casein kinase 1 enzymes (CK1) form a family of serine/threonine kinases with seven CK1 isoforms identified in humans. The most important substrates of CK1 kinases are proteins that act in the regulatory nodes essential for tumorigenesis of hematological malignancies. Among those, the most important are the functions of CK1s in the regulation of Wnt pathways, cell proliferation, apoptosis and autophagy. In this review we summarize the recent developments in the understanding of biology and therapeutic potential of the inhibition of CK1 isoforms in the pathogenesis of chronic lymphocytic leukemia (CLL), other non-Hodgkin lymphomas (NHL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and multiple myeloma (MM). CK1δ/ε inhibitors block CLL development in preclinical models via inhibition of WNT-5A/ROR1-driven non-canonical Wnt pathway. While no selective CK1 inhibitors have reached clinical stage to date, one dual PI3Kδ and CK1ε inhibitor, umbralisib, is currently in clinical trials for CLL and NHL patients. In MDS, AML and MM, inhibition of CK1α, acting via activation of p53 pathway, showed promising preclinical activities and the first CK1α inhibitor has now entered the clinical trials.     

Molecular Targeting of the Oncoprotein PLK1 in Pediatric Acute Myeloid Leukemia: RO3280, a Novel PLK1 Inhibitor,Induces Apoptosis in Leukemia Cells

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC₅₀) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC₅₀ of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.     

Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry

HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLA_peptide tetramer complexes (HLA-E_VMAPRTLIL, HLA-E_VMAPRSLLL, and HLA-A2_NLVPMVATV) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV⁻ and HCMV⁺ individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-E_UL40 and HLA-A2_pp65CD8 T cells, and expansion of γδT and NK subsets including δ2⁻γT and memory-like NKG2C⁺CD57⁺ NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines.     

Reversible Carbon-Carbon Bond Cleavage of a 3-Vinyl-1-Cyclopropene by Rh(I). Molecular Structures of Two Sterically Crowded 1,2,3,5-η-Pentadienediyl Complexes of Rh(III)

1,2,3-Tri-tert-butyl-3-vinyl-1-cyclopropene 6a reacts with [RhCl(C₂H₄)]₂ to give the dimeric 1,2,3,5-η-pentadienediyl complex 7a . The cyclopentadienyl derivative of this complex, 8a , is obtained by reaction of 7a with T1(C₅H₅). Crystal structures of both 7a and 8a were determined: 7a ; orthorhombic, Pbcn, a = 28.136 (4) Å, b = 10.637 (2) Å, c = 12.154 (2) Å, V = 3637.6 (1.2) ų, and Z = 4 : 8a ; triclinic, P 1 -bar, a = 9.906 (2) Å, b = 9.736 (2) Å, c = 12.126 (2) Å, α = 76.07 (2)°, β = 78.21 (2)°, γ = 65.34 (2)°, V = 1024.6 (3) ų, and Z = 2. Treatment of 7a with one equivalent of PMe₃ per Rh center results in regeneration of the vinylcyclopropene, demonstrating the reversibility of the ring opening reaction. Deuterium labelling studies show that both the ring opening and closing reactions proceed with retention of configuration at the vinyl olefin. Thus the trans-deuterated vinylcyclopropene 6b reacts with [RhCl(C₂H₄)₂]₂ to give 7b in which deuterium is located exclusively in the syn position. Treatment of 7b with T1(C₅H₅) yields 8b . Treatment of 7b with one equivalent of PMe₃ per Rh center results in formation of 6b as the sole organic product. Unlike their triphenyl relative 2 , the tri-Bu complexes 7a , 7b, 8a , and 8b are stereochemically rigid in solution at room temperature, and do not undergo an η³ → η¹ → η³ isomerization on the NMR time scale. However, after prolonged heating, syn-anti site exchange of H and D slowly takes place with 8b .     

Effects of Aflatoxin B1 on T-Cell Subsets and mRNA Expression of Cytokines in the Intestine of Broilers

This study was conducted to investigate the effects of aflatoxin B₁ (AFB₁) on T-cell subsets and mRNA expression of cytokines in the small intestine of broilers. One hundred and fifty-six one-day-old healthy Cobb broilers were randomly divided into control group (0 mg/kg AFB₁) and AFB₁ group (0.6 mg/kg AFB₁) with three replicates per group and 26 birds per replicate for 21 days, respectively. At 7, 14, and 21 days of age, the duodenum, jejunum and ileum were sampled for analyzing T cell subsets (CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺) by flow cytometry as well as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α mRNA expression by qRT-PCR. The percentages of T-cells in the intra-epithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) of duodenum, jejunum and ileum in the AFB₁ group showed a decreased tendency in comparison to the control group. The mRNA expression of cytokines in the three intestinal segments in the AFB₁ group presented a general decline compared with the control groups. Our data demonstrated that 0.6 mg/kg AFB₁ in the broilers diet could reduce the percentages of T-cell subsets and the expression level of cytokine mRNA in the small intestine, implying that the immune function of the intestinal mucosa might be affected. The reduction of cytokines mRNA expression may be closely associated with the decreased proportions of T cells subsets induced by AFB₁.     

Development and Structural Evaluation of N-Alkylated trans-2-Phenylcyclopropylamine-Based LSD1 Inhibitors

Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the demethylation of histone H3 and regulates gene expression. Because it is implicated in the regulation of diseases such as acute myeloid leukemia, potent LSD1-specific inhibitors have been pursued. Trans-2-phenylcyclopropylamine (2-PCPA)-based inhibitors featuring substitutions on the amino group have emerged, with sub-micromolar affinities toward LSD1 and high selectivities over monoamine oxidases (MAOs). We synthesized two N-alkylated 2-PCPA-based LSD1 inhibitors, S2116 and S2157, based on the previously developed S2101. S2116 and S2157 exhibited enhanced potency for LSD1 by 2.0- to 2.6-fold, as compared with S2101. In addition, they exhibited improved selectivity over MAOs. Structural analyses of LSD1 co-crystallized with S2101, S2116, S2157, or another N-alkylated inhibitor (FCPA-MPE) confirmed that the N-substituents enhance the potency of a 2-PCPA-based inhibitor of LSD1, without constituting the adduct formed with FAD.     

Novel Very Long-Chain α-Methoxylated Δ5,9 Fatty Acids from the Sponge Asteropus niger Are Effective Inhibitors of Topoisomerases IB

The novel fatty acids (2R,5Z,9Z)‐2‐methoxy‐25‐methyl‐5,9‐hexacosadienoic acid ( 1a ) and (2R,5Z,9Z)‐2‐methoxy‐24‐methyl‐5,9‐hexacosadienoic acid ( 1b ) were isolated in 80 % purity from the Caribbean sponge Asteropus niger by chloroform/methanol extraction followed by solvent partitioning and silica gel column chromatography. The compounds were characterized by utilizing a combination of gas chromatography‐mass spectrometry, nuclear magnetic resonance, and circular dichroism. Acids 1a and 1b were not detected in the phospholipids (PtdCho and PtdIns) of the sponge, but rather as free FA and possibly in glycosylceramides. The mixtures of 1a and 1b displayed cytotoxicity towards THP‐1 and HepG2 cells with EC_50's between 41 and 35 μg/mL. Apoptosis was not the preferred mode of cell death induced by 1a – 1b in the THP‐1 cells. This implies other types of cytotoxicity mechanisms, such as membrane disruption and/or the inhibition (EC₅₀ = 1.8 μg/mL) of the human topoisomerase IB enzyme (hTopIB), with a mechanism of inhibition different from the one displayed by camptothecin (CPT). In a separate experiment, the mixture of 1a and 1b also displayed cytotoxicity towards ex vivo mouse splenocytes infected with Leishmania infantum amastigotes (IC₅₀ = 0.17 mg/mL) and free living promastigotes (IC₅₀ = 0.34 mg/mL). It was also found that the FA were inhibitory of the Leishmania topoisomerase IB (LTopIB) with an EC₅₀ = 5.1 μg/mL. Taken together, 1a and 1b represent a new class of FA with potential as TopIB inhibitors that preferentially inhibit hTopIB over LTopIB.     

Preparation and some spectroscopic properties ofN-heterocyclic derivatives of a novel 1-pyrroline C18 fatty ester

A C₁₈ 1-pyrroline fatty ester, methyl 8-(5-hexyl-2-pyrrolin-1-yl)octanoate (1), was prepared from methyliso-ricinoleate. The C=N bond of the pyrroline ring was oxidized bym-chloroperoxy-benzoic acid to yield a mixture of oxaziridine isomers 2a,2b, which decomposed during gas chromatographic analysis to a 2,5-disubstituted pyrrole derivative, methyl 8-(5-hexyl-1H-pyrrole-2-)octanoate (3). Compound 3 was also obtained by reaction of 2a,2b with dilute HCl in methanol. Reaction of compound 1 with iodo-methane formed anN-methyl iminium iodide intermediate 4, which on reduction with sodium borohydride furnished a mixture ofcis/trans-N-methyl-2,5-disubstituted pyrrolidine derivatives, methyl 8-(cis/trans-5-hexyl-N-methyl-pyrrolidine-2-)octanoates 5a,5b. Reduction of compound 1 with NaBH₄ gave a mixture ofcis/trans-isomers of 2,5-disubstituted pyrrolidine derivatives, methyl 8-(5-hexyl-pyrrolidine-2-)octanoates 6a,6b. Acetylation of compounds 6a,6b with acetic anhydride furnished the correspondingN-acetyl pyrrolidines 7a,7b. When compound 1 was treated with perchloric acid, the corresponding iminium perchlorate derivative, methyl 8-(5-hexyl-1-pyrrolinium perchlorate-2-)octanoate 8 was obtained. The structures of the various derivatives were characterized by a combination of chromatographic, mass spectral and spectroscopic techniques.     

3,5‐Bis(benzylidene)‐4‐oxo‐1‐phosphonopiperidines and Related Diethyl Esters: Potent Cytotoxins with Multi‐Drug‐Resistance Reverting Properties

A series of 3,5‐bis(benzylidene)‐4‐piperidones 3 were converted into the corresponding 3,5‐bis(benzylidene)‐1‐phosphono‐4‐piperidones 5 via diethyl esters 4 . The analogues in series 4 and 5 displayed marked growth inhibitory properties toward human Molt 4/C8 and CEM T‐lymphocytes as well as murine leukemia L1210 cells. In general, the N‐phosphono compounds 5 , which are more hydrophilic than the analogues in series 3 and 4 , were the most potent cluster of cytotoxins, and, in particular, 3,5‐bis‐(2‐nitrobenzylidene)‐1‐phosphono‐4‐piperidone 5 g had an average IC₅₀ value of 34 nM toward the two T‐lymphocyte cell lines. Four of the compounds displayed potent cytotoxicity toward a panel of nearly 60 human tumor cell lines, and nanomolar IC₅₀ values were observed in a number of cases. The mode of action of 5 g includes the induction of apoptosis and inhibition of cellular respiration. Most of the members of series 4 as well as several analogues in series 5 are potent multi‐drug resistance (MDR) reverting compounds. Various correlations were noted between certain molecular features of series 4 and 5 and cytotoxic properties, affording some guidelines in expanding this study.     

Structure refinement of the as-synthesized and the calcined form of zeolite RUB-3 (RTE)

The structure analysis based on single crystal and powder data revealed that the framework structure of RUB-3 (structure type code RTE) consists of small [4⁴5⁴6²] building units and medium–large [4⁶5⁴6⁶8²] cages with a free volume of ca. 300 ų. Interconnected cages form a one-dimensional channel system with narrow, slightly elliptical pore openings (free diameter 3.6×4.3 Ų). ¹H–¹³C CP MAS NMR spectroscopy proved that the cages are occupied by disordered (±)-exo-2-aminobicyclo[2.2.1]heptane molecules which were used as templates during the synthesis. The unit cell composition of as-synthesized RUB-3 is (C₇H₁₃N)₂·[Si₂₄O₄₈]. The lattice parameters, bond lengths, bond angles and unit cell volumes are nearly identical for the as-synthesized [a=14.039(2) Å, b=13.602(2) Å, c=7.428(1) Å, β=102.22(3)°] and calcined form [a=14.018(1) Å, b=13.612(1) Å, c=7.418(1) Å, β=102.12(1)°] of RUB-3. The RTE structure even keeps its symmetry (C2/m) during the calcination process, indicating that the RTE framework topology is very rigid. All crystals are four-fold twins with twin planes {110}.     

Inflammation in the Human Periodontium Induces Downregulation of the α1- and β1-Subunits of the sGC in Cementoclasts

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α₁- and β₁-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α₁- and β₁-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α₁- and β₁-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.     

Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca²⁺ imaging, and computational modeling, we evaluated the effects of S1P on the Ca²⁺-activated K⁺ currents (I_K(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BK_Ca channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca²⁺ concentration (Ca_i) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BK_Ca was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed I_K(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of I_K(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced I_K(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BK_Ca conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited I_K(Ca) and the permissive role of S1P on the BK_Ca activity was also effectively observed in the PC12 cell system. The S1P-mediated I_K(Ca) stimulation may result from the elevated Ca_i, whereas the inhibition of BK_Ca activity by S1P appears to be direct. By the differentiated tailoring BK_Ca channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.     

Distinct Immune Signatures Indicative of Treatment Response and Immune-Related Adverse Events in Melanoma Patients under Immune Checkpoint Inhibitor Therapy

To identify potential early biomarkers of treatment response and immune-related adverse events (irAE), a pilot immune monitoring study was performed in stage IV melanoma patients by flow cytometric analysis of peripheral blood mononuclear cells (PBMC). Overall, 17 patients were treated with either nivolumab or pembrolizumab alone, or with a combination of nivolumab and ipilimumab every three weeks. Of 15 patients for which complete response assessment was available, treatment responders (n = 10) as compared to non-responders (n = 5) were characterized by enhanced PD-1 expression on CD8⁺ T cells immediately before treatment (median ± median absolute deviation/MAD 26.7 ± 10.4% vs. 17.2 ± 5.3%). Responders showed a higher T cell responsiveness after T cell receptor ex vivo stimulation as determined by measurement of programmed cell death 1 (PD-1) expression on CD3⁺ T cells before the second cycle of treatment. The percentage of CD8⁺ effector memory (CD8⁺CD45RA⁻CD45RO⁺CCR7⁻) T cells was higher in responders compared to non-responders before and immediately after the first cycle of treatment (median ± MAD 39.2 ± 7.3% vs. 30.5 ± 4.1% and 37.7 ± 4.6 vs. 24.0 ± 6.4). Immune-related adverse events (irAE) were accompanied by a higher percentage of activated CD4⁺ (CD4⁺CD38⁺HLADR⁺) T cells before the second treatment cycle (median ± MAD 14.9 ± 3.9% vs. 5.3 ± 0.4%). In summary, PBMC immune monitoring of immune-checkpoint inhibition (ICI) treatment in melanoma appears to be a promising approach to identify early markers of treatment response and irAEs.     

LSD1小分子抑制剂的合成及抗肿瘤活性研究

设计并合成了一系列以苯甲酰肼类结构为母核的赖氨酸特异性组蛋白去甲基化酶1(LSD1)小分子抑制剂,并研究其体外抗肿瘤活性。首先,通过体外酶水平单浓度抑制实验进行了初步评价,并随后进一步考察目标化合物对多种LSD1高表达肿瘤细胞株增殖的抑制作用,化合物结构经质谱及核磁共振表征确证。活性评价结果显示,3-(((3R,5S)-3,5-二甲基吗啉代)磺酰基)-N'-(7-羟基-2,3-二氢-1H-茚-1-亚基)苯甲酰肼、N'-(1-(5-氯-2-羟基苯基)亚乙基)-3-(((3R,5S)-3,5-二甲基吗啉代)磺酰基)苯甲酰肼和N'-(4-氯-7-羟基-2,3-二氢-1H-茚-1-亚基)-3-((4-吗啉代哌啶-1-基)磺酰基)苯甲酰肼可显著抑制肿瘤细胞的增殖,并有4个目标化合物对体外多种LSD1高表达的肿瘤细胞株增殖有抑制作用,其中3-(((3R,5S)-3,5-二甲基吗啉代)磺酰基)-N'-(7-羟基-2,3-二氢-1H-茚-1-亚基)苯甲酰肼对BGC823、HCT116、A2780s的半数抑制浓度分别为0.32、0.54、0.90μmol/L。     

Hypoxia-Inducible Factor-2 Alpha Regulates the Migration of Fibroblast-like Synoviocytes via Oxidative Stress-Induced CD70 Expression in Patients with Rheumatoid Arthritis

This study aimed to examine the role of CD70, which is highly expressed on fibroblast-like synoviocytes (FLS), in rheumatoid arthritis (RA) patients. FLS isolated from RA (n = 14) and osteoarthritis (OA, n = 4) patients were stimulated with recombinant interleukin-17 (IL-17; 5 ng/mL) and tumor necrosis factor alpha (TNF-α; 5 ng/mL) for 24 h. Expression of CD70, CD27/soluble CD27 (sCD27), and hypoxia-inducible factor-2 alpha (HIF-2α) was analyzed by RT-qPCR, flow cytometry, and ELISA assays, respectively. Reactive oxygen species (ROS) expression and cell migration were also examined. The HIF-2α inhibitor PT-2385 and CD70 inhibitor BU69 were used to specifically suppress these pathways. Stimulation with IL-17 and TNF-α significantly induced CD70 expression in RA FLS. Although the synovial fluids from patients with RA contained high levels of sCD27, surface expression of CD27, a ligand of CD70, was rarely detected in RA FLS. Cytokine-induced CD70 expression was significantly decreased following antioxidant treatment. Following HIF-2α inhibition, RA FLS had decreased expression of CD70 and ROS levels. Migration of RA FLS was also inhibited by inhibition of CD70 or HIF-2α. The surface expression of CD70 is regulated by HIF-2α and ROS levels and is a key contributor to cytokine-enhanced migration in RA FLS.     

Developing an Irreversible Inhibitor of Human DDAH‐1, an Enzyme Upregulated in Melanoma

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase‐1 (DDAH‐1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. In this study, N⁵‐(1‐imino‐2‐chloroethyl)‐L ‐ornithine (Cl‐NIO) is shown to be a potent time‐ and concentration‐dependent inhibitor of purified human DDAH‐1 (K_I=1.3±0.6 μM ; k_inact=0.34±0.07 min^?1), with >500‐fold selectivity against two arginine‐handling enzymes in the same pathway. An activity probe is used to measure the “in cell” IC₅₀ value (6.6±0.2 μM ) for Cl‐NIO inhibition of DDAH‐1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50/64 (78 %) of melanoma lines tested showed increased levels of DDAH‐1 relative to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl‐NIO shows a subsequent decrease in cellular nitric oxide production. Cl‐NIO is a promising tool for the study of methylarginine‐mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production, such as septic shock and idiopathic pulmonary fibrosis.     

A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML

HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.