Enhancing personalized immune checkpoint therapy by immune archetyping and pharmacological targeting.

Immune checkpoint inhibitors (ICIs) are an expanding class of immunotherapeutic agents with the potential to cure cancer. Despite the outstanding clinical response in patient subsets, most individuals become refractory or develop resistance. Patient stratification and personalized immunotherapies are limited by the absence of predictive response markers. Recent findings show that dominant patterns of immune cell composition, T-cell status and heterogeneity, and spatiotemporal distribution of immune cells within the tumor microenvironment (TME) are becoming essential determinants of prognosis and therapeutic response. In this context, ICIs also function as investigational tools and proof of concept, allowing the validation of the identified mechanisms. After reviewing the current state of ICIs, this article will explore new comprehensive predictive markers for ICIs based on recent discoveries. We will discuss the recent establishment of a classification of TMEs into immune archetypes as a tool for personalized immune profiling, allowing patient stratification before ICI treatment. We will discuss the developing comprehension of T-cell diversity and its role in shaping the immune profile of patients. We describe the potential of strategies that score the mutual spatiotemporal modulation between T-cells and other cellular components of the TME. Additionally, we will provide an overview of a range of synthetic and naturally occurring or derived small molecules. We will compare compounds that were recently identified by in silico prediction to wet lab-validated drug candidates with the potential to function as ICIs and/or modulators of the cellular components of the TME.

Marine Polyether Phycotoxin Palytoxin Induces Apoptotic Cell Death via Mcl-1 and Bcl-2 Downregulation.

Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.

Anti-Leukemic Properties of Aplysinopsin Derivative EE-84 Alone and Combined to BH3 Mimetic A-1210477.

Aplysinopsins are a class of marine indole alkaloids that exhibit a wide range of biological activities. Although both the indole and N-benzyl moieties of aplysinopsins are known to possess antiproliferative activity against cancer cells, their mechanism of action remains unclear. Through in vitro and in vivo proliferation and viability screening of newly synthesized aplysinopsin analogs on myelogenous leukemia cell lines and zebrafish toxicity tests, as well as analysis of differential toxicity in noncancerous RPMI 1788 cells and PBMCs, we identified EE-84 as a promising novel drug candidate against chronic myeloid leukemia. This indole derivative demonstrated drug-likeness in agreement with Lipinski's rule of five. Furthermore, EE-84 induced a senescent-like phenotype in K562 cells in line with its cytostatic effect. EE-84-treated K562 cells underwent morphological changes in line with mitochondrial dysfunction concomitant with autophagy and ER stress induction. Finally, we demonstrated the synergistic cytotoxic effect of EE-84 with a BH3 mimetic, the Mcl-1 inhibitor A-1210477, against imatinib-sensitive and resistant K562 cells, highlighting the inhibition of antiapoptotic Bcl-2 proteins as a promising novel senolytic approach against chronic myeloid leukemia.

Petromurin C Induces Protective Autophagy and Apoptosis in FLT3-ITD-Positive AML: Synergy with Gilteritinib.

Treatment of acute myeloid leukemia (AML) remains inefficient due to drug resistance and relapse, particularly in patients with FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD). Marine-derived natural products have recently been used for drug development against AML. We show in this study that petromurin C, which was isolated from the culture extract of the marine-derived fungus Aspergillus candidus KUFA0062, isolated from the marine sponge Epipolasis sp., induces early autophagy followed by apoptotic cell death via activation of the intrinsic cell death pathway concomitant with mitochondrial stress and downregulation of Mcl-1 in FLT3-ITD mutated MV4-11 cells. Moreover, petromurin C synergized with the clinically-used FLT3 inhibitor gilteritinib at sub-toxic concentrations. Altogether, our results provide preliminary indications that petromurin C provides anti-leukemic effects alone or in combination with gilteritinib.

Non-canonical programmed cell death mechanisms triggered by natural compounds.

Natural compounds are the fundament of pharmacological treatments and more than 50% of all anticancer drugs are of natural origins or at least derived from scaffolds present in Nature. Over the last 25 years, molecular mechanisms triggered by natural anticancer compounds were investigated. Emerging research showed that molecules of natural origins are useful for both preventive and therapeutic purposes by targeting essential hallmarks and enabling characteristics described by Hanahan and Weinberg. Moreover, natural compounds were able to change the differentiation status of selected cell types. One of the earliest response of cells treated by pharmacologically active compounds is the change of its morphology leading to ultra-structural perturbations: changes in membrane composition, cytoskeleton integrity, alterations of the endoplasmic reticulum, mitochondria and of the nucleus lead to formation of morphological alterations that are a characteristic of both compound and cancer type preceding cell death. Apoptosis and autophagy were traditionally considered as the most prominent cell death or cell death-related mechanisms. By now multiple other cell death modalities were described and most likely involved in response to chemotherapeutic treatment. It can be hypothesized that especially necrosis-related phenotypes triggered by various treatments or evolving from apoptotic or autophagic mechanisms, provide a more efficient therapeutic outcome depending on cancer type and genetic phenotype of the patient. In fact, the recent discovery of multiple regulated forms of necrosis and the initial elucidation of the corresponding cell signaling pathways appear nowadays as important tools to clarify the immunogenic potential of non-canonical forms of cell death induction.

Anticancer and Immunogenic Properties of Cardiac Glycosides.

Cardiac glycosides (CGs) are natural compounds widely used in the treatment of several cardiac conditions and more recently have been recognized as potential antitumor compounds. They are known to be ligands for Na/K-ATPase, which is a promising drug target in cancer. More recently, in addition to their antitumor effects, it has been suggested that CGs activate tumor-specific immune responses. This review summarizes the anticancer aspects of CGs as new strategies for immunotherapy and drug repositioning (new horizons for old players), and the possible new targets for CGs in cancer cells.

2,5-Dimethyl-celecoxib inhibits cell cycle progression and induces apoptosis in human leukemia cells.

Cyclooxygenase-2 (COX-2) is an essential regulator of cancer promotion and progression. Extensive efforts to target this enzyme have been developed to reduce growth of cancer cells for chemopreventive and therapeutic reasons. In this context, cyclooxygenase-2 inhibitors present interesting antitumor effects. However, inhibition of COX-2 by anti-COX-2 compounds such as celecoxib was recently associated with detrimental cardiovascular side effects limiting their clinical use. As many anticancer effects of celecoxib are COX-2 independent, analogs such as 2,5-dimethyl-celecoxib (DMC), which lacks COX-2-inhibitory activity, represent a promising alternative strategy. In this study, we investigated the effect of this molecule on growth of hematologic cancer cell lines (U937, Jurkat, Hel, Raji, and K562). We found that this molecule is able to reduce the growth and induces apoptosis more efficiently than celecoxib in all the leukemic cell lines tested. Cell death was associated with downregulation of Mcl-1 protein expression. We also found that DMC induces endoplasmic reticulum stress, which is associated with a decreased of GRP78 protein expression and an alteration of cell cycle progression at the G1/S transition in U937 cells. Accordingly, typical downregulation of c-Myc and cyclin D1 and an upregulation of p27 were observed. Interestingly, for shorter time points, an alteration of mitotic progression, associated with the downregulation of survivin protein expression was observed. Altogether, our data provide new evidence about the mode of action of this compound on hematologic malignancies.

Celecoxib prevents curcumin-induced apoptosis in a hematopoietic cancer cell model.

Molecules targeting pro-inflammatory pathways have demonstrated beneficial effects in cancer treatment. More recently, combination of natural and synthetic anti-inflammatory drugs was suggested as an appealing strategy to inhibit tumor growth. Herein, we show that curcumin, a polyphenol from Curcuma longa and celecoxib induce apoptosis in hematopoietic cancer cell lines (Hel, Jurkat, K562, Raji, and U937). Further investigations on the most sensitive cell line, U937, indicated that these effects were tightly associated with an accumulation of the cells in S and G2/M for curcumin and in G0/G1 phase of cell cycle for celecoxib, respectively. The effect of celecoxib on cell cycle is associated with an induction of p27 and the down-regulation of cyclin D1. However, in the case of combination experiments, the pretreatment of U937 cells with celecoxib at non-apoptogenic concentrations counteracted curcumin-induced apoptosis. We found that this effect correlated with the prevention of the accumulation in S and G2/M phase of cell cycle induced by curcumin. Similar results have been obtained when celecoxib and curcumin were co-administrated at the same time. Overall our data suggest that this natural and synthetic drug combination is detrimental for cell death induction.

A novel coumarin-quinone derivative SV37 inhibits CDC25 phosphatases, impairs proliferation, and induces cell death.

Cell division cycle (CDC) 25 proteins are key phosphatases regulating cell cycle transition and proliferation by regulating CDK/cyclin complexes. Overexpression of these enzymes is frequently observed in cancer and is related to aggressiveness, high-grade tumors and poor prognosis. Thus, targeting CDC25 by compounds, able to inhibit their activity, appears a good therapeutic approach. Here, we describe the synthesis of a new inhibitor (SV37) whose structure is based on both coumarin and quinone moieties. An analytical in vitro approach shows that this compound efficiently inhibits all three purified human CDC25 isoforms (IC50 1-9 µM) in a mixed-type mode. Moreover, SV37 inhibits growth of breast cancer cell lines. In MDA-MB-231 cells, reactive oxygen species generation is followed by pCDK accumulation, a mark of CDC25 dysfunction. Eventually, SV37 treatment leads to activation of apoptosis and DNA cleavage, underlining the potential of this new type of coumarin-quinone structure.

PPARγ-inactive Δ2-troglitazone independently triggers ER stress and apoptosis in breast cancer cells.

Our aim was to better understand peroxisome proliferator-activated receptor gamma (PPARγ)-independent pathways involved in anti-cancer effects of thiazolidinediones (TZDs). We focused on Δ2-troglitazone (Δ2-TGZ), a PPARγ inactive TZD that affects breast cancer cell viability. Appearance of TUNEL positive cells, changes in mitochondrial membrane potential, cleavage of poly(ADP-ribose) polymerase (PARP)-1 and caspase-7 revealed that apoptosis occurred in both hormone-dependent MCF7 and hormone-independent MDA-MB-231 breast cancer cells after 24 and 48 h of treatment. A microarray study identified endoplasmic reticulum (ER) stress as an essential cellular function since many genes involved in ER stress were upregulated in MCF7 cells following Δ2-TGZ treatment. Δ2-TGZ-induced ER stress was further confirmed in MCF7 cells by phosphorylation of pancreatic endoplasmic reticulum kinase-like endoplasmic reticulum kinase (PERK) and its target eIF2α after 1.5 h, rapid increase in activating transcription factor (ATF) 3 mRNA levels, splicing of X-box binding protein 1 (XBP1) after 3 h, accumulation of binding immunogloblulin protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) after 6 h. Immunofluorescence microscopy indicated that CHOP was relocalized to the nucleus of treated cells. Similarly, in MDA-MB-231 cells, overexpression of ATF3, splicing of XBP1, and accumulation of BiP and CHOP were observed following Δ2-TGZ treatment. In MCF7 cells, knock-down of CHOP or the inhibition of c-Jun N-terminal kinase (JNK) did not impair cleavage of PARP-1 and caspase-7. Altogether, our results show that ER stress is an early response of major types of breast cancer cells to Δ2-TGZ, prior to, but not causative of apoptosis.

Tanzawaic acids isolated from a marine-derived fungus of the genus Penicillium with cytotoxic activities.

Tanzawaic acids M (1), N (2), O (3) and P (4) and the known tanzawaic acids B (5) and E (6), have been isolated from an extract of a cultured marine-derived fungus (strain CF07370) identified as a member of the genus Penicillium. The structures of 1-4 were determined based on spectroscopic evidence. The antimicrobial and cytotoxic activities of compounds 1-6 were evaluated.

A Survey of Marine Natural Compounds and Their Derivatives with Anti-cancer Activity Reported in 2012.

Although considerable effort and progress has been made in the search for new anticancer drugs and treatments in the last several decades, cancer remains a major public health problem and one of the major causes of death worldwide. Many sources, including plants, animals, and minerals, are of interest in cancer research because of the possibility of identifying novel molecular therapeutics. Moreover, structure-activity-relationship (SAR) investigations have become a common way to develop naturally derived or semi-synthetic molecular analogues with improved efficacy and decreased toxicity. In 2012, approximately 138 molecules from marine sources, including isolated compounds and their associated analogues, were shown to be promising anticancer drugs. Among these, 62% are novel compounds. In this report, we review the marine compounds identified in 2012 that may serve as novel anticancer drugs.

Plumbagin modulates leukemia cell redox status.

Plumbagin is a plant naphtoquinone exerting anti-cancer properties including apoptotic cell death induction and generation of reactive oxygen species (ROS). The aim of this study was to elucidate parameters explaining the differential leukemia cell sensitivity towards this compound. Among several leukemia cell lines, U937 monocytic leukemia cells appeared more sensitive to plumbagin treatment in terms of cytotoxicity and level of apoptotic cell death compared to more resistant Raji Burkitt lymphoma cells. Moreover, U937 cells exhibited a ten-fold higher ROS production compared to Raji. Neither differential incorporation, nor efflux of plumbagin was detected. Pre-treatment with thiol-containing antioxidants prevented ROS production and subsequent induction of cell death by apoptosis whereas non-thiol-containing antioxidants remained ineffective in both cellular models. We conclude that the anticancer potential of plumbagin is driven by pro-oxidant activities related to the cellular thiolstat.

ATP1A1/BCL2L1 predicts the response of myelomonocytic and monocytic acute myeloid leukemia to cardiac glycosides.

Myelomonocytic and monocytic acute myeloid leukemia (AML) subtypes are intrinsically resistant to venetoclax-based regimens. Identifying targetable vulnerabilities would limit resistance and relapse. We previously documented the synergism of venetoclax and cardiac glycoside (CG) combination in AML. Despite preclinical evidence, the repurposing of cardiac glycosides (CGs) in cancer therapy remained unsuccessful due to a lack of predictive biomarkers. We report that the ex vivo response of AML patient blasts and the in vitro sensitivity of established cell lines to the hemi-synthetic CG UNBS1450 correlates with the ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1)/BCL2 like 1 (BCL2L1) expression ratio. Publicly available AML datasets identify myelomonocytic/monocytic differentiation as the most robust prognostic feature, along with core-binding factor subunit beta (CBFB), lysine methyltransferase 2A (KMT2A) rearrangements, and missense Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Mechanistically, BCL2L1 protects from cell death commitment induced by the CG-mediated stepwise triggering of ionic perturbation, protein synthesis inhibition, and MCL1 downregulation. In vivo, CGs showed an overall tolerable profile while impacting tumor growth with an effect ranging from tumor growth inhibition to regression. These findings suggest a predictive marker for CG repurposing in specific AML subtypes.

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation.

BACKGROUND: Despite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence. METHODS: Transcriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice. RESULTS: Transcriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients' stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities. CONCLUSIONS: Overall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

A survey of marine natural compounds and their derivatives with anti-cancer activity reported in 2011.

Cancer continues to be a major public health problem despite the efforts that have been made in the search for novel drugs and treatments. The current sources sought for the discovery of new molecules are plants, animals and minerals. During the past decade, the search for anticancer agents of marine origin to fight chemo-resistance has increased greatly. Each year, several novel anticancer molecules are isolated from marine organisms and represent a renewed hope for cancer therapy. The study of structure-function relationships has allowed synthesis of analogues with increased efficacy and less toxicity. In this report, we aim to review 42 compounds of marine origin and their derivatives that were published in 2011 as promising anticancer compounds.

ROS-independent JNK activation and multisite phosphorylation of Bcl-2 link diallyl tetrasulfide-induced mitotic arrest to apoptosis.

Garlic-derived organosulfur compounds including diallyl polysulfides are well known for various health-beneficial properties and recent reports even point to a potential role of diallyl polysulfides as chemopreventive and therapeutic agents in cancer treatment due to their selective antiproliferative effects. In this respect, diallyl tri- and tetrasulfide are reported as strong inducers of an early mitotic arrest and subsequent apoptosis, but the underlying molecular mechanisms and the link between these two events are not yet fully elucidated. Our data revealed that diallyl tetrasulfide acts independently of reactive oxygen species and tubulin represents one of its major cellular targets. Tubulin depolymerization prevents the formation of normal spindle microtubules, thereby leading to G2/M arrest. Here, we provide evidence that c-jun N-terminal kinase, which is activated early in response to diallyl tetrasulfide treatment, mediates multisite phosphorylation and subsequent proteolysis of the anti-apoptotic protein B-cell lymphoma 2. As the latter event occurs concomitantly with the onset of apoptosis and the chemical c-jun N-terminal kinase inhibitor SP600125 not only prevented B-cell lymphoma 2 phosphorylation and proteolysis but also apoptosis following diallyl tetrasulfide treatment, we suggest that these c-jun N-terminal kinase-mediated modulations of B-cell lymphoma 2 represent the missing link connecting early microtubule inactivation to the induction of apoptosis.

Phytochemical Screening and Antioxidant and Cytotoxic Effects of Acacia macrostachya.

Acacia macrostachya is used in Burkina Faso folk medicine for the treatment of inflammation and cancer. The purpose of this study was to evaluate the antioxidant and cytotoxic effects of this plant. The cytotoxic effects of root (dichloromethane B1 and methanol B2) and stem (dichloromethane B3 and methanol B4) bark extracts of A. macrostachya were assessed on chronic K562 and acute U937 myeloid leukemia cancer cells using trypan blue, Hoechst, and MitoTracker Red staining methods. The antioxidant content of extracts was evaluated using DPPH (2,2-diphenyl-1-picryl-hydrazyl) and FRAP (ferric reducing antioxidant power) methods. The root bark extracts B1 and B2 of A. macrostachya demonstrated higher cytotoxicity with IC50 values in a low µg/mL range on both U937 and K562 cells, while the stem bark B4 extract selectively affected U937 cells. Overall, healthy proliferating peripheral blood mononuclear cells (pPBMCs) were not or barely impacted in the range of concentrations cytotoxic to cancer cells. In addition, A. macrostachya exhibited significant antioxidant content with 646.06 and 428.08 µg ET/mg of extract for the B4 and B2 extracts, respectively. Phytochemical screening showed the presence of flavonoids, tannins, alkaloids, and terpenoids/steroids. The results of this study highlight the interest of A. macrostachya extracts for the isolation of anticancer molecules.

BH3 Mimetics in AML Therapy: Death and Beyond?

B cell lymphoma 2 (BCL2) homology domain 3 (BH3) mimetics are targeted therapeutic agents that allow response prediction and patient stratification. BH3 mimetics are prototypical activators of the mitochondrial death program in cancer. They emerged as important modulators of cellular mechanisms contributing to poor therapeutic responses, including cancer cell stemness, cancer-specific metabolic routes, paracrine signaling to the tumor microenvironment, and immune modulation. We present an overview of the antagonism between BH3 mimetics and antiapoptotic BCL2 proteins. We focus on acute myeloid leukemia (AML), a cancer with reduced therapeutic options that have recently been improved by BH3 mimetics.

Tetrahydrobenzimidazole TMQ0153 triggers apoptosis, autophagy and necroptosis crosstalk in chronic myeloid leukemia.

By comparing imatinib-sensitive and -resistant chronic myeloid leukemia (CML) cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the capacity of this compound to release immunogenic cell death (ICD) markers. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.