Discovery of 5-{2-[5-Chloro-2-(5-ethoxyquinoline-8-sulfonamido)phenyl]ethynyl}-4-methoxypyridine-2-carboxylic Acid, a Highly Selective in Vivo Useable Chemical Probe to Dissect MCT4 Biology.

Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.

Release of adenosine-induced immunosuppression: Comprehensive characterization of dual A2A/A2B receptor antagonist.

Immunosuppression is one of the main mechanisms facilitating tumor expansion. It may be driven by immune checkpoint protein expression, anti-inflammatory cytokine secretion or enhanced metabolic enzyme production, leading to the subsequent build-up of metabolites such as adenosine. Under physiological conditions, adenosine prevents the development of tissue damage resulting from a prolonged immune response; the same mechanism might be employed by tumor tissue to promote immunosuppression. Immune cells expressing A2A and A2B adenosine receptors present in an adenosine-rich environment have suppressed effector functions, such as cytotoxicity, proinflammatory cytokine release, antigen presentation and others, making them inert to cancer cells. This study was designed to investigate the dual antagonist potential of SEL330-639 to abolish adenosine-driven immunosuppression. SEL330-639 has slow dissociation kinetics. It inhibits cAMP production in human CD4+ cells, CD8+ cells and moDCs, which leads to diminished CREB phosphorylation and restoration of antitumor cytokine production (IL-2, TNFα, IL-12) in multiple primary human immune cells. The aforementioned results were additionally validated by gene expression analysis and functional assays in which NK cell line cytotoxicity was recovered by SEL330-639. Adenosine-driven immunosuppression is believed to preclude the effectiveness of immune checkpoint inhibitor therapies. Hence, there is an urgent need to develop new immuno-oncological strategies. Here, we comprehensively characterize SEL330-639, a novel dual A2A/A2B receptor antagonist effective in both lymphoid and myeloid cell populations with nanomolar potency. Due to its tight binding to the A2A and A2B receptors, this binding is sustained even at high adenosine concentrations mimicking the upper limit of the range of adenosine levels observed in the tumor microenvironment.

Discovery of a series of ester-substituted NLRP3 inflammasome inhibitors.

The NLRP3 inflammasome is a component of the innate immune system involved in the production of proinflammatory cytokines. Aberrant activation by a wide range of exogenous and endogenous signals can lead to chronic, low-grade inflammation. It has attracted a great deal of interest as a drug target due to the association with diseases of large unmet medical need such as Alzheimer's disease, Parkinson's disease, arthritis, and cancer. To date, no drugs specifically targeting inhibition of the NLRP3 inflammasome have been approved. In this work, we used the known NLRP3 inflammasome inhibitor CP-456,773 (aka CRID3 or MCC 950) as our starting point and undertook a Structure-Activity Relationship (SAR) analysis and subsequent scaffold-hopping exercise. This resulted in the rational design of a series of novel ester-substituted urea compounds that are highly potent and selective NLRP3 inflammasome inhibitors, as exemplified by compounds 44 and 45. It is hypothesized that the ester moiety acts as a highly permeable delivery vehicle and is subsequently hydrolyzed to the carboxylic acid active species by carboxylesterase enzymes. These molecules are greatly differentiated from the state-of-the-art and offer potential in the treatment of NLRP3-driven diseases, particularly where tissue penetration is required.

Synthetically Lethal Interactions of Heme Oxygenase-1 and Fumarate Hydratase Genes.

Elevated expression of heme oxygenase-1 (HO-1, encoded by HMOX1) is observed in various types of tumors. Hence, it is suggested that HO-1 may serve as a potential target in anticancer therapies. A novel approach to inhibit HO-1 is related to the synthetic lethality of this enzyme and fumarate hydratase (FH). In the current study, we aimed to validate the effect of genetic and pharmacological inhibition of HO-1 in cells isolated from patients suffering from hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-an inherited cancer syndrome, caused by FH deficiency. Initially, we confirmed that UOK 262, UOK 268, and NCCFH1 cell lines are characterized by non-active FH enzyme, high expression of Nrf2 transcription factor-regulated genes, including HMOX1 and attenuated oxidative phosphorylation. Later, we demonstrated that shRNA-mediated genetic inhibition of HMOX1 resulted in diminished viability and proliferation of cancer cells. Chemical inhibition of HO activity using commercially available inhibitors, zinc and tin metalloporphyrins as well as recently described new imidazole-based compounds, especially SLV-11199, led to decreased cancer cell viability and clonogenic potential. In conclusion, the current study points out the possible relevance of HO-1 inhibition as a potential anti-cancer treatment in HLRCC. However, further studies revealing the molecular mechanisms are still needed.

Development and characterization of a new inhibitor of heme oxygenase activity for cancer treatment.

Heme oxygenase-1 (HO-1, HMOX1) degrades pro-oxidant heme into carbon monoxide (CO), ferrous ions (Fe2+) and biliverdin. The enzyme exerts multiple cytoprotective functions associated with the promotion of angiogenesis and counteraction of the detrimental effects of cellular stress which are crucial for the survival of both normal and tumor cells. Accordingly, in many tumor types, high expression of HO-1 correlates with poor prognosis and resistance to treatment, i.e. chemotherapy, suggesting inhibition of HO-1 as a possible antitumor approach. At the same time, the lack of selective and well-profiled inhibitors of HO-1 determines the unmet need for new modulators of this enzyme, with the potential to be used in either adjuvant therapy or as the stand-alone targeted therapeutics. In the current study, we provided novel inhibitors of HO-1 and validated the effect of pharmacological inhibition of HO activity by the imidazole-based inhibitor (SLV-11199) in human pancreatic (PANC-1) and prostate (DU-145) cancer cell lines. We demonstrated potent inhibition of HO activity in vitro and showed associated anticancer effectiveness of SLV-11199. Treatment with the tested compound led to decreased cancer cell viability and clonogenic potential. It has also sensitized the cancer cells to chemotherapy. In PANC-1 cells, diminished HO activity resulted in down-regulation of pro-angiogenic factors like IL-8. Mechanistic investigations revealed that the treatment with SLV-11199 decreased cell migration and inhibited MMP-1 and MMP-9 expression. Moreover, it affected mesenchymal phenotype by regulating key modulators of the epithelial to mesenchymal transition (EMT) signalling axis. Finally, F-actin cytoskeleton and focal contacts were destabilized by the reported compound. Overall, the current study suggests a possible relevance of the tested novel inhibitor of HO activity as a potential anticancer compound. To support such utility, further investigation is still needed, especially in in vivo conditions.

Microenvironment-induced PIM kinases promote CXCR4-triggered mTOR pathway required for chronic lymphocytic leukaemia cell migration.

Lymph node microenvironment provides chronic lymphocytic leukaemia (CLL) cells with signals promoting their survival and granting resistance to chemotherapeutics. CLL cells overexpress PIM kinases, which regulate apoptosis, cell cycle and migration. We demonstrate that BCR crosslinking, CD40 stimulation, and coculture with stromal cells increases PIMs expression in CLL cells, indicating microenvironment-dependent PIMs regulation. PIM1 and PIM2 expression at diagnosis was higher in patients with advanced disease (Binet C vs. Binet A/B) and in those, who progressed after first-line treatment. In primary CLL cells, inhibition of PIM kinases with a pan-PIM inhibitor, SEL24-B489, decreased PIM-specific substrate phosphorylation and induced dose-dependent apoptosis in leukaemic, but not in normal B cells. Cytotoxicity of SEL24-B489 was similar in TP53-mutant and TP53 wild-type cells. Finally, inhibition of PIM kinases decreased CXCR4-mediated cell chemotaxis in two related mechanisms-by decreasing CXCR4 phosphorylation and surface expression, and by limiting CXCR4-triggered mTOR pathway activity. Importantly, PIM and mTOR inhibitors similarly impaired migration, indicating that CXCL12-triggered mTOR is required for CLL cell chemotaxis. Given the microenvironment-modulated PIM expression, their pro-survival function and a role of PIMs in CXCR4-induced migration, inhibition of these kinases might override microenvironmental protection and be an attractive therapeutic strategy in this disease.

A novel, dual pan-PIM/FLT3 inhibitor SEL24 exhibits broad therapeutic potential in acute myeloid leukemia.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common genetic lesions in acute myeloid leukemia patients (AML). Although FLT3 tyrosine kinase inhibitors initially exhibit clinical activity, resistance to treatment inevitably occurs within months. PIM kinases are thought to be major drivers of the resistance phenotype and their inhibition in relapsed samples restores cell sensitivity to FLT3 inhibitors. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. For such reasons, we have developed SEL24-B489 - a potent, dual PIM and FLT3-ITD inhibitor. SEL24-B489 exhibited significantly broader on-target activity in AML cell lines and primary AML blasts than selective FLT3-ITD or PIM inhibitors. SEL24-B489 also demonstrated marked activity in cells bearing FLT3 tyrosine kinase domain (TKD) mutations that lead to FLT3 inhibitor resistance. Moreover, SEL24-B489 inhibited the growth of a broad panel of AML cell lines in xenograft models with a clear pharmacodynamic-pharmacokinetic relationship. Taken together, our data highlight the unique dual activity of the SEL24-B489 that abrogates the activity of signaling circuits involved in proliferation, inhibition of apoptosis and protein translation/metabolism. These results underscore the therapeutic potential of the dual PIM/FLT3-ITD inhibitor for the treatment of AML.

Expression of PIM kinases in Reed-Sternberg cells fosters immune privilege and tumor cell survival in Hodgkin lymphoma.

Reed-Sternberg (RS) cells of classical Hodgkin lymphoma (cHL) express multiple immunoregulatory proteins that shape the cHL microenvironment and allow tumor cells to evade immune surveillance. Expression of certain immunoregulatory proteins is modulated by prosurvival transcription factors, such as NFκB and STATs. Because these factors also induce expression of the oncogenic PIM1/2/3 serine/threonine kinases, and as PIMs modulate transcriptional activity of NFκB and STATs, we hypothesized that these kinases support RS cell survival and foster their immune privilege. Here, we investigated PIM1/2/3 expression in cHL and assessed their role in developing RS cell immune privilege and survival. PIM1/2/3 were ubiquitously expressed in primary and cultured RS cells, and their expression was driven by JAK-STAT and NFκB activity. Genetic or chemical PIM inhibition with a newly developed pan-PIM inhibitor, SEL24-B489, induced RS cell apoptosis. PIM inhibition decreased cap-dependent protein translation, blocked JAK-STAT signaling, and markedly attenuated NFκB-dependent gene expression. In a cHL xenograft model, SEL24-B489 delayed tumor growth by 95.8% (P = .0002). Furthermore, SEL24-B489 decreased the expression of multiple molecules engaged in developing the immunosuppressive microenvironment, including galectin-1 and PD-L1/2. In coculture experiments, T cells incubated with SEL24-B489-treated RS cells exhibited higher expression of activation markers than T cells coincubated with control RS cells. Taken together, our data indicate that PIM kinases in cHL exhibit pleiotropic effects, orchestrating tumor immune escape and supporting RS cell survival. Inhibition of PIM kinases decreases RS cell viability and disrupts signaling circuits that link these cells with their niches. Thus, PIM kinases are promising therapeutic targets in cHL.