Transcription Factor Activation Profiles (TFAP) identify compounds promoting differentiation of Acute Myeloid Leukemia cell lines

Repurposing of drugs for new therapeutic use has received considerable attention for its potential to limit time and cost of drug development. Here we present a new strategy to identify chemicals that are likely to promote a desired phenotype. We used data from the Connectivity Map (CMap) to produce a ranked list of drugs according to their potential to activate transcription factors that mediate myeloid differentiation of leukemic progenitor cells. To validate our strategy, we tested the in vitro differentiation potential of candidate compounds using the HL-60 human cell line as a myeloid differentiation model. Ten out of 22 compounds, which were ranked high in the inferred list, were confirmed to promote significant differentiation of HL-60. These compounds may be considered candidate for differentiation therapy. The method that we have developed is versatile and it can be adapted to different drug repurposing projects.

Insights on Cancer Cell Inhibition, Subcellular Activities, and Kinase Profile of Phenylacetamides Pending 1H-Imidazol-5-One Variants

Structural changes of small-molecule drugs may bring interesting biological properties, especially in the field of kinase inhibitors. We sought to study tirbanibulin, a first-in-class dual Src kinase (non-ATP competitive)/tubulin inhibitor because there was not enough reporting about its structure–activity relationships (SARs). In particular, the present research is based on the replacement of the outer ring of the biphenyl system of 2-[(1,1′-biphenyl)-4-yl]-N-benzylacetamide, the identified pharmacophore of KX chemotype, with a heterocyclic ring. The newly synthesized compounds showed a range of activities in cell-based anticancer assays, agreeing with a clear SAR profile. The most potent compound, (Z)-N-benzyl-4-[4-(4-methoxybenzylidene)-2-methyl-5-oxo-4,5-dihydro-1H-imidazol-1-yl]phenylacetamide (KIM-161), demonstrated cytotoxic IC50 values at 294 and 362 nM against HCT116 colon cancer and HL60 leukemia cell lines, respectively. Profiling of this compound (aqueous solubility, liver microsomal stability, cytochrome P450 inhibition, reactivity with reduced glutathione, and plasma protein binding) confirmed its adequate drug-like properties. Mechanistic studies revealed that this compound does not depend on tubulin or Src kinase inhibition as a factor in forcing HL60 to exit its cell cycle and undergo apoptosis. Instead, KIM-161 downregulated several other kinases such as members of BRK, FLT, and JAK families. It also strongly suppresses signals of ERK1/2, GSK-3α/β, HSP27, and STAT2, while it downregulated AMPKα1 phosphorylation within the HL60 cells. Collectively, these results suggest that phenylacetamide-1H-imidazol-5-one (KIM-161) could be a promising lead compound for further clinical anticancer drug development.

In Vitro Anticancer Activity and Mechanism of Action of an Aziridinyl Galactopyranoside

We recently screened a series of new aziridines β-D-galactopyranoside derivatives for selective anticancer activity and identified 2-methyl-2,3-[N-(4-methylbenzenesulfonyl)imino]propyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D-galactopyranoside (AzGalp) as the most promising compound. In this article, we explore the possible mechanisms involved in the cytotoxicity of this aziridine and evaluate its selective anticancer activity using cancer cells and normal cells from a variety of tissues. Our data show that AzGalp induces DNA damage (comet assay). Cells deficient in the nucleotide excision repair (NER) pathway were hypersensitive to the cytotoxicity of this compound. These results suggest that AzGalp induces bulky DNA adducts, and that cancer cells lacking a functional NER pathway may be particularly vulnerable to the anticancer effects of this aziridine. Several experiments revealed that neither the generation of oxidative stress nor the inhibition of glycolysis played a significant role in the cytotoxicity of AzGalp. Combinations of AzGalp with oxaliplatin or 5-fluorouracil slightly improved the ability of both anticancer drugs to selectively kill cancer cells. AzGalp also showed selective cytotoxicity against a panel of malignant cells versus normal cells; the highest selectivity was observed for two acute promyelocytic leukemia cell lines. Additional preclinical studies are necessary to evaluate the anticancer potential of AzGalp.

Discovery of Sulforaphane as an Inducer of Ferroptosis in U-937 Leukemia Cells: Expanding Its Anticancer Potential

In recent years, natural compounds have emerged as inducers of non-canonical cell death. The isothiocyanate sulforaphane (SFN) is a well-known natural anticancer compound with remarkable pro-apoptotic activity. Its ability to promote non-apoptotic cell-death mechanisms remains poorly investigated. This work aimed to explore the capacity of SFN to induce non-apoptotic cell death modalities. SFN was tested on different acute myeloid leukemia cell lines. The mechanism of cell death was investigated using a multi-parametric approach including fluorescence microscopy, western blotting, and flow cytometry. SFN triggered different cell-death modalities in a dose-dependent manner. At 25 μM, SFN induced caspase-dependent apoptosis and at 50 μM ferroptosis was induced through depletion of glutathione (GSH), decreased GSH peroxidase 4 protein expression, and lipid peroxidation. In contrast, necroptosis was not involved in SFN-induced cell death, as demonstrated by the non-significant increase in phosphorylation of receptor-interacting protein kinase 3 and phosphorylation of the necroptotic effector mixed lineage kinase domain-like pseudokinase. Taken together, our results suggest that the antileukemic activity of SFN can be mediated via both ferroptotic and apoptotic cell death modalities.

Clinically Relevant Oxygraphic Assay to Assess Mitochondrial Energy Metabolism in Acute Myeloid Leukemia Patients

Resistant acute myeloid leukemia (AML) exhibits mitochondrial energy metabolism changes compared to newly diagnosed AML. This phenotype is often observed by evaluating the mitochondrial oxygen consumption of blasts, but most of the oximetry protocols were established from leukemia cell lines without validation on primary leukemia cells. Moreover, the cultures and storage conditions of blasts freshly extracted from patient blood or bone marrow cause stress, which must be evaluated before determining oxidative phosphorylation (OXPHOS). Herein, we evaluated different conditions to measure the oxygen consumption of blasts using extracellular flow analyzers. We first determined the minimum number of blasts required to measure OXPHOS. Next, we compared the OXPHOS of blasts cultured for 3 h and 18 h after collection and found that to maintain metabolic organization for 18 h, cytokine supplementation is necessary. Cytokines are also needed when measuring OXPHOS in cryopreserved, thawed and recultured blasts. Next, the concentrations of respiratory chain inhibitors and uncoupler FCCP were established. We found that the FCCP concentration required to reach the maximal respiration of blasts varied depending on the patient sample analyzed. These protocols provided can be used in future clinical studies to evaluate OXPHOS as a biomarker and assess the efficacy of treatments targeting mitochondria.

In vitro cytotoxic and leishmanicidal activity of isolated and semisynthetic ent-pimaranes from Aldama arenaria

Two pimaranes ent-pimara -8(14),15-dien-19-oic acid (1) and ent-8(14),15-pimaradien-3β-ol (2), isolated from Aldama arenaria, and six semi-synthetic derivatives methyl ester of the ent-pimara-8(14),15-dien-19-oic acid (3), ent-pimara-8(14),15-dien-19-ol (4), acetate of ent-pimara-8(14),15-dien-19-ol (5), ent-pimara-8(14),15-dien-19-ol succinic acid (6), acetate of ent-8(14),15-pimaradien-3β-ol (7), ent-8(14),15-pimaradien-3β-ol succinic acid (8) were evaluated in vitro for their cytotoxic activities to childhood leukemia cell lines and leishmanicidal activity against the parasite Leishmania amazonensis. Among these compounds, 1 to 6 presented moderate cytotoxic activity, with compound 4 being the most active (GI50 of 2.6 µM for the HL60 line) and the derivatives 7 and 8 inactive. Against the parasite Leishmania amazonensis, the most promising derivative was acetate of ent-pimara-8(14),15-dien-19-ol (5), with EC50 of 20.1 µM, selectivity index of 14.3, and significant reduction in the parasite load. Pimarane analogues 1, ent-pimara-8(14),15-dien-19-oic acid, and 2, ent-8(14),15-pimaradien-3β-ol, presented different activities, corroborating the application of such molecules as prototypes for the design of other derivatives that have greater cytotoxic or leishmanicidal potential.

Modeling, Synthesis and Biological Evaluation of Potential Retinoid-X-Receptor (RXR) Selective Agonists: Analogs of 4-[1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahyro-2-naphthyl)ethynyl]benzoic Acid (Bexarotene) and 6-(Ethyl(4-isobutoxy-3-isopropylphenyl)amino)nicotinic Acid (NEt-4IB)

Five novel analogs of 6-(ethyl)(4-isobutoxy-3-isopropylphenyl)amino)nicotinic acid—or NEt-4IB—in addition to seven novel analogs of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene) were prepared and evaluated for selective retinoid-X-receptor (RXR) agonism alongside bexarotene (1), a FDA-approved drug for cutaneous T-cell lymphoma (CTCL). Bexarotene treatment elicits side-effects by provoking or disrupting other RXR-dependent pathways. Analogs were assessed by the modeling of binding to RXR and then evaluated in a human cell-based RXR-RXR mammalian-2-hybrid (M2H) system as well as a RXRE-controlled transcriptional system. The analogs were also tested in KMT2A-MLLT3 leukemia cells and the EC50 and IC50 values were determined for these compounds. Moreover, the analogs were assessed for activation of LXR in an LXRE system as drivers of ApoE expression and subsequent use as potential therapeutics in neurodegenerative disorders, and the results revealed that these compounds exerted a range of differential LXR-RXR activation and selectivity. Furthermore, several of the novel analogs in this study exhibited reduced RARE cross-signaling, implying RXR selectivity. These results demonstrate that modification of partial agonists such as NEt-4IB and potent rexinoids such as bexarotene can lead to compounds with improved RXR selectivity, decreased cross-signaling of other RXR-dependent nuclear receptors, increased LXRE-heterodimer selectivity, and enhanced anti-proliferative potential in leukemia cell lines compared to therapeutics such as 1.

Development of Astatine-211 (211At)-Based Anti-CD123 Radioimmunotherapy for Acute Leukemias and Other CD123+ Hematologic Malignancies

Background: Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia. Of current interest are alpha-particle emitting radionuclides as they deliver a very large amount of radiation over just a few cell diameters, enabling efficient and selective target cell kill. So far, alpha-emitters including astatine-211 (211At) have been primarily explored with monoclonal antibodies (mAbs) targeting CD45 or CD33 but their broad display on non-malignant target-expressing cells can lead to marked "on-target, off tumor cell" toxicities. To overcome this limitation, we developed a novel form of 211At-based RIT targeting CD123. CD123 is displayed widely on acute leukemia cells, including underlying leukemic stem cells, but is expressed only on a discrete subset of normal hematopoietic cells and is virtually absent on non-blood cells. Methods: We immunized BALB/c mice with peptides consisting of the extracellular domain of human CD123 to generate anti-CD123 mAbs. Flow cytometry-based assays with human acute leukemia cell lines were used to characterize binding of hybridoma supernatants and mAbs to CD123. mAbs were conjugated with isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10), a boron cage molecule for subsequent astatination, and were then labeled with 211At. In vivo leukemia cell targeting ("biodistribution") and efficacy studies were conducted in immunodeficient NOD-Rag1 null IL2rɣ null/J (NRG) mice xenografted with MOLM-13 cells, a CD123+ human acute myeloid leukemia cell line. Results: Based on initial hybridoma screening studies, we selected 4 mAbs (10C4, 5G4, 11F11, and 1H8) for further characterization. Phenotyping studies with CD123+ and CD123- human acute leukemia cell lines (including CD123+ cell lines in which CD123 was deleted via CRISPR/Cas9) confirmed specific binding of all mAbs to human CD123 (binding intensity: 10C4>5G4=11F11=1H8), with 10C4 yielding a higher median fluorescence intensity than the widely used commercial anti-CD123 mAb clones, 7G3 and 6H6 (Figure 1). In vitro internalization with a panel of human acute leukemia cell lines studies demonstrated uptake of all mAbs by CD123+ target cells with a kinetic slower than that for anti-CD33 antibodies (typically, 30-50% of the anti-CD123 mAb internalized over 2-4 hours). All 4 anti-CD123 mAbs could be conjugated to B10 and subsequently labeled with 211At. Unlike a non-binding 211At-labeled control mAb, 211At-labeled anti-CD123 mAbs showed uptake at MOLM-13 flank tumors in NRG mice carrying MOLM-13 xenografts. After additional leukemia cell targeting studies to optimize the dosing of 10C4, we conducted proof-of-concept efficacy studies in NRG mice injected intravenously with luciferase-transduced MOLM-13 cells (disseminated leukemia model). Animals were either untreated or treated with 10 µCi, 20 µCi, or 40 µCi of 211At-labeled 10C4-B10 mAb (9-11 animals/group). This was followed by the infusion of bone marrow cells from donor mice as stem cell support 3 days later. As shown in Figure 2 and Figure 3, 211At-10C4-B10 led to a dose dependent decrease in tumor burden. Further, the treatment significantly prolonged survival compared to untreated animals (median survival: 49 days [40 µCi of 211At] vs. 31 days [10 µCi of 211At] vs. 21 days [Ctrl]; P<0.0001 for Ctrl vs. 10 µCi, P<0.004 for 10 µCi vs. 40 µCi), demonstrating potent in vivo anti-leukemia efficacy of a single dose of 211At-CD123 RIT. Conclusion: Our data support the further development of 211At-CD123 RIT for the treatment of patients with acute leukemia and other CD123+ hematologic malignancies. Figure 1 Figure 1. Disclosures Green: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Cellectar Biosciences: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; JANSSEN Biotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Patents & Royalties, Research Funding; Legend Biotech: Consultancy; Neoleukin Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; SpringWorks Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.