Quinoxaline derivatives: Recent discoveries and development strategies towards anticancer agents.

Cancer is a leading cause of death and a major health problem worldwide. While many effective anticancer agents are available, most drugs currently on the market are not specific, raising issues like the common side effects of chemotherapy. However, recent research hold promises for the development of more efficient and safer anticancer drugs. Quinoxaline and its derivatives are becoming recognized as a novel class of chemotherapeutic agents with activity against different tumors. The present review compiles and discusses studies concerning the therapeutic potential of the anticancer activity of quinoxaline derivatives, covering articles published between January 2018 and January 2023.

Combination drug screen targeting glioblastoma core vulnerabilities reveals pharmacological synergisms.

BACKGROUND: Pharmacological synergisms are an attractive anticancer strategy. However, with more than 5000 approved-drugs and compounds in clinical development, identifying synergistic treatments represents a major challenge. METHODS: High-throughput screening was combined with target deconvolution and functional genomics to reveal targetable vulnerabilities in glioblastoma. The role of the top gene hit was investigated by RNA interference, transcriptomics and immunohistochemistry in glioblastoma patient samples. Drug combination screen using a custom-made library of 88 compounds in association with six inhibitors of the identified glioblastoma vulnerabilities was performed to unveil pharmacological synergisms. Glioblastoma 3D spheroid, organotypic ex vivo and syngeneic orthotopic mouse models were used to validate synergistic treatments. FINDINGS: Nine targetable vulnerabilities were identified in glioblastoma and the top gene hit RRM1 was validated as an independent prognostic factor. The associations of CHK1/MEK and AURKA/BET inhibitors were identified as the most potent amongst 528 tested pairwise drug combinations and their efficacy was validated in 3D spheroid models. The high synergism of AURKA/BET dual inhibition was confirmed in ex vivo and in vivo glioblastoma models, without detectable toxicity. INTERPRETATION: Our work provides strong pre-clinical evidence of the efficacy of AURKA/BET inhibitor combination in glioblastoma and opens new therapeutic avenues for this unmet medical need. Besides, we established the proof-of-concept of a stepwise approach aiming at exploiting drug poly-pharmacology to unveil druggable cancer vulnerabilities and to fast-track the identification of synergistic combinations against refractory cancers. FUNDING: This study was funded by institutional grants and charities.

Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors.

Brain tumors are an important cause of suffering and death. Glioblastoma are the most frequent primary tumors of the central nervous system in adults. They are associated with a very poor prognosis, since only 10% of GBM patients survive 5 years after diagnosis. Medulloblastoma are the most frequent brain malignancies in childhood; they affect the cerebellum in children under 10 years of age in 75% of cases. The current multimodal treatment comes at the expense of serious and often long-lasting side effects. Herein, we propose the synthesis of a library of novel alkoxyamines as anticancer drug candidates. The most efficient molecule, ALK4, was selected based on its ability to inhibit both survival and migration of GBM and MB cells in 2D cultures and in 3D tumor spheroids. A fluorescent derivative was used to show the early cytosolic accumulation of ALK4 in tumor cells. Spontaneous homolysis of ALK4 led to the release of alkyl radicals, which triggered the generation of reactive oxygen species, fragmentation of the mitochondrial network and ultimately apoptosis. To control its homolytic process, the selected alkoxyamine was bioconjugated to a peptide selectively recognized by matrix metalloproteases. This bioconjugate, named ALK4-MMPp, successfully inhibited survival, proliferation, and invasion of GBM and MB tumor micromasses. We further developed innovative brain and cerebellum organotypic models to monitor treatment response over time. It confirmed that ALK4-MMPp significantly impaired tumor progression, while no significant damage was observed on normal brain tissue. Lastly, we showed that ALK4-MMPp was well-tolerated in vivo by zebrafish embryos. This study provides a new strategy to control the activation of alkoxyamines, and revealed the bioconjugate ALK4-MMPp bioconjugate as a good anticancer drug candidate.

Targeting of the ELR+CXCL/CXCR1/2 Pathway Is a Relevant Strategy for the Treatment of Paediatric Medulloblastomas.

Medulloblastoma (MB) is the most common and aggressive paediatric brain tumour. Although the cure rate can be as high as 70%, current treatments (surgery, radio- and chemotherapy) excessively affect the patients' quality of life. Relapses cannot be controlled by conventional or targeted treatments and are usually fatal. The strong heterogeneity of the disease (four subgroups and several subtypes) is related to innate or acquired resistance to reference treatments. Therefore, more efficient and less-toxic therapies are needed. Here, we demonstrated the efficacy of a novel inhibitor (C29) of CXCR1/2 receptors for ELR+CXCL cytokines for the treatment of childhood MB. The correlation between ELR+CXCL/CXCR1/2 expression and patient survival was determined using the R2: Genomics Analysis and Visualization platform. In vitro efficacy of C29 was evaluated by its ability to inhibit proliferation, migration, invasion, and pseudo-vessel formation of MB cell lines sensitive or resistant to radiotherapy. The growth of experimental MB obtained by MB spheroids on organotypic mouse cerebellar slices was also assayed. ELR+CXCL/CXCR1/2 levels correlated with shorter survival. C29 inhibited proliferation, clone formation, CXCL8/CXCR1/2-dependent migration, invasion, and pseudo-vessel formation by sensitive and radioresistant MB cells. C29 reduced experimental growth of MB in the ex vivo organotypic mouse model and crossed the blood-brain barrier. Targeting CXCR1/2 represents a promising therapeutic strategy for the treatment of paediatric MB in first-line treatment or after relapse following conventional therapy.

Beta-blockers disrupt mitochondrial bioenergetics and increase radiotherapy efficacy independently of beta-adrenergic receptors in medulloblastoma.

BACKGROUND: Medulloblastoma is the most frequent brain malignancy of childhood. The current multimodal treatment comes at the expense of serious and often long-lasting side effects. Drug repurposing is a strategy to fast-track anti-cancer therapy with low toxicity. Here, we showed the ability of ß-blockers to potentiate radiotherapy in medulloblastoma with bad prognosis. METHODS: Medulloblastoma cell lines, patient-derived xenograft cells, 3D spheroids and an innovative cerebellar organotypic model were used to identify synergistic interactions between ß-blockers and ionising radiations. Gene expression profiles of ß-adrenergic receptors were analysed in medulloblastoma samples from 240 patients. Signaling pathways were explored by RT-qPCR, RNA interference, western blotting and RNA sequencing. Medulloblastoma cell bioenergetics were evaluated by measuring the oxygen consumption rate, the extracellular acidification rate and superoxide production. FINDINGS: Low concentrations of ß-blockers significantly potentiated clinically relevant radiation protocols. Although patient biopsies showed detectable expression of ß-adrenergic receptors, the ability of the repurposed drugs to potentiate ionising radiations did not result from the inhibition of the canonical signaling pathway. We highlighted that the efficacy of the combinatorial treatment relied on a metabolic catastrophe that deprives medulloblastoma cells of their adaptive bioenergetics capacities. This led to an overproduction of superoxide radicals and ultimately to an increase in ionising radiations-mediated DNA damages. INTERPRETATION: These data provide the evidence of the efficacy of ß-blockers as potentiators of radiotherapy in medulloblastoma, which may help improve the treatment and quality of life of children with high-risk brain tumours. FUNDING: This study was funded by institutional grants and charities.

Metronomic Chemotherapy Modulates Clonal Interactions to Prevent Drug Resistance in Non-Small Cell Lung Cancer.

Despite recent advances in deciphering cancer drug resistance mechanisms, relapse is a widely observed phenomenon in advanced cancers, mainly due to intratumor clonal heterogeneity. How tumor clones progress and impact each other remains elusive. In this study, we developed 2D and 3D non-small cell lung cancer co-culture systems and defined a phenomenological mathematical model to better understand clone dynamics. Our results demonstrated that the drug-sensitive clones inhibit the proliferation of the drug-resistant ones under untreated conditions. Model predictions and their experimental in vitro and in vivo validations indicated that a metronomic schedule leads to a better regulation of tumor cell heterogeneity over time than a maximum-tolerated dose schedule, while achieving control of tumor progression. We finally showed that drug-sensitive and -resistant clones exhibited different metabolic statuses that could be involved in controlling the intratumor heterogeneity dynamics. Our data suggested that the glycolytic activity of drug-sensitive clones could play a major role in inhibiting the drug-resistant clone proliferation. Altogether, these computational and experimental approaches provide foundations for using metronomic therapy to control drug-sensitive and -resistant clone balance and highlight the potential of targeting cell metabolism to manage intratumor heterogeneity.

Tumor uptake and associated greater efficacy of anti-Her2 immunoliposome does not rely on Her2 expression status: study of a docetaxel-trastuzumab immunoliposome on Her2+ breast cancer model (SKBR3).

Nanoparticles have been used for decades in breast cancer. More recently, anti-human epidermal receptor 2 (Her2) immunoliposomes are of rising interest. However, recent studies have questioned the actual relevance of using anti-Her2 antibodies to improve liposome distribution and efficacy. Using standard thin-film method and maleimide linker, we have synthesized a 140-nm docetaxel-trastuzumab immunoliposome. This nanoparticle was then tested on a canonical Her2-overexpressing breast cancer model (i.e., SKBR3), using 3D spheroids and xenografted mice. Its efficacy was compared with free docetaxel + trastuzumab, liposomal docetaxel + free trastuzumab and to reference antibody-drug conjugate trastuzumab-emtansine (T-DM1). Immunoliposomes resulted in better efficacy as compared with all other treatments, both in vitro and in vivo. To explain such an improvement, immunoliposome biodistribution was investigated using live imaging in xenografted mice. Surprisingly, no difference in tumor uptake was found between anti-Her2 immunoliposomes and standard docetaxel liposomes (i.e., 1.9 ± 1.2 vs. 1.7 ± 0.5% at the end of treatment and 1.4 ± 0.6 vs. 1.6 ± 0.4% at the end of the study, respectively, P > 0.05). We hypothesized that passive targeting (i.e., enhanced permeation and retention effect) contributed more to tumor distribution than active targeting and that the observed differences in efficacy could come from a better internalization of immunoliposomes into Her2+ cells as compared with standard liposomes, and not from a higher specificity towards tumor tissue.

Chemical modifications of imidazole-containing alkoxyamines increase C-ON bond homolysis rate: Effects on their cytotoxic properties in glioblastoma cells.

Previously, we described alkoxyamines bearing a pyridine ring as new pro-drugs with low molecular weights and theranostic activity. Upon chemical stimulus, alkoxyamines undergo homolysis and release free radicals, which can, reportedly, enhance magnetic resonance imaging and trigger cancer cell death. In the present study, we describe the synthesis and the anti-cancer activity of sixteen novel alkoxyamines that contain an imidazole ring. Activation of the homolysis was conducted by protonation and/or methylation. These new molecules displayed cytotoxic activities towards human glioblastoma cell lines, including the U251-MG cells that are highly resistant to the conventional chemotherapeutic agent Temozolomide. We further showed that the biological activities of the alkoxyamines were not only related to their half-life times of homolysis. We lastly identified the alkoxyamine (RS/SR)-4a, with both a high antitumour activity and favourable logD7.4 and pKa values, which make it a robust candidate for blood-brain barrier penetrating therapeutics against brain neoplasia.

From 3D spheroids to tumor bearing mice: efficacy and distribution studies of trastuzumab-docetaxel immunoliposome in breast cancer.

PURPOSE: Nanoparticles are of rising interest in cancer research, but in vitro canonical cell monolayer models are not suitable to evaluate their efficacy when prototyping candidates. Here, we developed three-dimensional (3D) spheroid models to test the efficacy of trastuzumab-docetaxel immunoliposomes in breast cancer prior to further testing them in vivo. MATERIALS AND METHODS: Immunoliposomes were synthesized using the standard thin film method and maleimide linker. Two human breast cancer cell lines varying in Her2 expression were tested: Her2+ cells derived from metastatic site: mammary breast MDA-MB-453 and triple-negative MDA-MB-231 cells. 3D spheroids were developed and tested with fluorescence detection to evaluate viability. In vivo efficacy and biodistribution studies were performed on xenograft bearing nude mice using fluorescent and bioluminescent imaging. RESULTS: In vitro, antiproliferative efficacy was dependent upon cell type, size of the spheroids, and treatment scheduling, resulting in subsequent changes between tested conditions and in vivo results. Immunoliposomes performed better than free docetaxel + free trastuzumab and ado-trastuzumab emtansine (T-DM1). On MDA-MB-453 and MDA-MB-231 cell growth was reduced by 76% and 25%, when compared to free docetaxel + free trastuzumab and by 85% and 70% when compared to T-DM1, respectively. In vivo studies showed tumor accumulation ranging from 3% up to 15% of the total administered dose in MDA-MB-453 and MDA-MB-231 bearing mice. When compared to free docetaxel + free trastuzumab, tumor growth was reduced by 89% (MDA-MB-453) and 25% (MDA-MB-231) and reduced by 66% (MDA-MB-453) and 29% (MDA-MB-231) when compared to T-DM1, an observation in line with data collected from 3D spheroids experiments. CONCLUSION: We demonstrated the predictivity of 3D in vitro models when developing and testing nanoparticles in experimental oncology. In vitro and in vivo data showed efficient drug delivery with higher efficacy and prolonged survival with immunoliposomes when compared to current anti-Her2 breast cancer strategies.

Metronomic Chemotherapy: Direct Targeting of Cancer Cells after all?

Metronomic chemotherapy (MC) was initially described as an antiangiogenic therapy more than 15 years ago. Over the past few years, additional data have highlighted the impact of MC on the microenvironment beyond angiogenesis, with, most importantly, a potential impact on the immune system. Here, we review and reappraise the fact that MC might be able to directly kill cancer cells. Although long neglected, this question is of critical importance both fundamentally and clinically, especially when considering future associations with immunotherapies.

Akt targeting as a strategy to boost chemotherapy efficacy in non-small cell lung cancer through metabolism suppression.

Metabolic reprogramming is a hallmark of cancer development, mediated by genetic and epigenetic alterations that may be pharmacologically targeted. Among oncogenes, the kinase Akt is commonly overexpressed in tumors and favors glycolysis, providing a rationale for using Akt inhibitors. Here, we addressed the question of whether and how inhibiting Akt activity could improve therapy of non-small cell lung cancer (NSCLC) that represents more than 80% of all lung cancer cases. First, we demonstrated that Akt inhibitors interacted synergistically with Microtubule-Targeting Agents (MTAs) and specifically in cancer cell lines, including those resistant to chemotherapy agents and anti-EGFR targeted therapies. In vivo, we further revealed that the chronic administration of low-doses of paclitaxel - i.e. metronomic scheduling - and the anti-Akt perifosine was the most efficient and the best tolerated treatment against NSCLC. Regarding drug mechanism of action, perifosine potentiated the pro-apoptotic effects of paclitaxel, independently of cell cycle arrest, and combining paclitaxel/perifosine resulted in a sustained suppression of glycolytic and mitochondrial metabolism. This study points out that targeting cancer cell bioenergetics may represent a novel therapeutic avenue in NSCLC, and provides a strong foundation for future clinical trials of metronomic MTAs combined with Akt inhibitors.

Metformin and propranolol combination prevents cancer progression and metastasis in different breast cancer models.

Discovery of new drugs for cancer treatment is an expensive and time-consuming process and the percentage of drugs reaching the clinic remains quite low.Drug repositioning refers to the identification and development of new uses for existing drugs and represents an alternative drug development strategy.In this work, we evaluated the antitumor effect of metronomic treatment with a combination of two repositioned drugs, metformin and propranolol, in triple negative breast cancer models.By in vitro studies with five different breast cancer derived cells, we observed that combined treatment decreased proliferation (P < 0.001), mitochondrial activity (P < 0.001), migration (P < 0.001) and invasion (P < 0.001). In vivo studies in immunocompetent mice confirmed the potential of this combination in reducing tumor growth (P < 0.001) and preventing metastasis (P < 0.05).Taken together our results suggest that metformin plus propranolol combined treatment might be beneficial for triple negative breast cancer control, with no symptoms of toxicity.

A Pathway Toward Tumor Cell-Selective CPPs?

Despite the great potential of CPPs in therapeutics and diagnosis, their application still suffers from a non-negligible drawback: a complete lack of cell-type specificity. In the innumerous routes proposed for CPP cell entry there is common agreement that electrostatic interactions between cationic CPPs and anionic components in membranes, including lipids and glycosaminoglycans, play a crucial role. Tumor cells have been shown to overexpress certain glycosaminoglycans at the cell membrane surface and to possess a higher amount of anionic lipids in their outer leaflet when compared with healthy cells. Such molecules confer tumor cell membranes an enhanced anionic character, a property that could be exploited by CPPs to preferentially target these cells. Herein, these aspects are discussed in an attempt to confer CPPs certain selectivity toward cancer cells.

Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo.

Glioblastoma is the most frequent primary brain tumor in adults. Because of molecular and cellular heterogeneity, high proliferation rate and significant invasive ability, prognosis of patients is poor. Recent therapeutic advances increased median overall survival but tumor recurrence remains inevitable. In this context, we used a high throughput screening approach to bring out novel compounds with anti-proliferative and anti-migratory properties for glioblastoma treatment. Screening of the Prestwick chemical library® of 1120 molecules identified proscillaridin A, a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase pump, with most significant effects on glioblastoma cell lines. In vitro effects of proscillaridin A were evaluated on GBM6 and GBM9 stem-like cell lines and on U87-MG and U251-MG cell lines. We showed that proscillaridin A displayed cytotoxic properties, triggered cell death, induced G2/M phase blockade in all the glioblastoma cell lines and impaired GBM stem self-renewal capacity even at low concentrations. Heterotopic and orthotopic xenotransplantations were used to confirm in vivo anticancer effects of proscillaridin A that both controls xenograft growth and improves mice survival. Altogether, results suggest that proscillaridin A is a promising candidate as cancer therapies in glioblastoma. This sustains previous reports showing that cardiac glycosides act as anticancer drugs in other cancers.

Metronomics: towards personalized chemotherapy?

Since its inception in 2000, metronomic chemotherapy has undergone major advances as an antiangiogenic therapy. The discovery of the pro-immune properties of chemotherapy and its direct effects on cancer cells has established the intrinsic multitargeted nature of this therapeutic approach. The past 10 years have seen a marked rise in clinical trials of metronomic chemotherapy, and it is increasingly combined in the clinic with conventional treatments, such as maximum-tolerated dose chemotherapy and radiotherapy, as well as with novel therapeutic strategies, such as drug repositioning, targeted agents and immunotherapy. We review the latest advances in understanding the complex mechanisms of action of metronomic chemotherapy, and the recently identified factors associated with disease resistance. We comprehensively discuss the latest clinical data obtained from studies performed in both adult and paediatric populations, and highlight ongoing clinical trials. In this Review, we foresee the future developments of metronomic chemotherapy and specifically its potential role in the era of personalized medicine.

ROS-mediated EB1 phosphorylation through Akt/GSK3ß pathway: implication in cancer cell response to microtubule-targeting agents.

Microtubule-targeting agents (MTAs) are largely administered in adults and children cancers. Better deciphering their mechanism of action is of prime importance to develop more convenient therapy strategies. Here, we addressed the question of how reactive oxygen species (ROS) generation by mitochondria can be necessary for MTA efficacy. We showed for the first time that EB1 associates with microtubules in a phosphorylation-dependent manner, under control of ROS. By using phospho-defective mutants, we further characterized the Serine 155 residue as critical for EB1 accumulation at microtubule plus-ends, and both cancer cell migration and proliferation. Phosphorylation of EB1 on the Threonine 166 residue triggered opposite effects, and was identified as a requisite molecular switch in MTA activities. We then showed that GSK3ß activation was responsible for MTA-triggered EB1 phosphorylation, resulting from ROS-mediated inhibition of upstream Akt. We thus disclosed here a novel pathway by which generation of mitochondrial ROS modulates microtubule dynamics through phosphorylation of EB1, improving our fundamental knowledge about this oncogenic protein, and pointing out the need to re-examine the current dogma of microtubule targeting by MTAs. The present work also provides a strong mechanistic rational to the promising therapeutic strategies that currently combine MTAs with anti-Akt targeted therapies.

A proapoptotic peptide conjugated to penetratin selectively inhibits tumor cell growth.

The peptide KLA (acetyl-(KLAKLAK)2-NH2), which is rather non toxic for eukaryotic cell lines, becomes active when coupled to the cell penetrating peptide, penetratin (Pen), by a disulfide bridge. Remarkably, the conjugate KLA-Pen is cytotoxic, at low micromolar concentrations, against a panel of seven human tumor cell lines of various tissue origins, including cells resistant to conventional chemotherapy agents but not to normal human cell lines. Live microscopy on cells possessing fluorescent labeled mitochondria shows that in tumor cells, KLA-Pen had a strong impact on mitochondria tubular organization instantly resulting in their aggregation, while the unconjugated KLA and pen peptides had no effect. But, mitochondria in various normal cells were not affected by KLA-Pen. The interaction with membrane models of KLA-Pen, KLA and penetratin were studied using dynamic light scattering, calorimetry, plasmon resonance, circular dichroism and ATR-FTIR to unveil the mode of action of the conjugate. To understand the selectivity of the conjugate towards tumor cell lines and its action on mitochondria, lipid model systems composed of zwitterionic lipids were used as mimics of normal cell membranes and anionic lipids as mimics of tumor cell and mitochondria membrane. A very distinct mode of interaction with the two model systems was observed. KLA-Pen may exert its deleterious and selective action on cancer cells by the formation of pores with an oblique membrane orientation and establishment of important hydrophobic interactions. These results suggest that KLA-Pen could be a lead compound for the design of cancer therapeutics.

Evidence for new targets and synergistic effect of metronomic celecoxib/fluvastatin combination in pilocytic astrocytoma.

BACKGROUND: Pilocytic astrocytomas occur predominantly in childhood. In contrast to the posterior fossa location, hypothalamo-chiasmatic pilocytic astrocytomas display a worse prognosis often leading to multiple surgical procedures and/or several lines of chemotherapy and radiotherapy to achieve long-term control. Hypothalamo-chiasmatic pilocytic astrocytomas and cerebellar pilocytic astrocytomas have a distinctive gene signature and several differential expressed genes (ICAM1, CRK, CD36, and IQGAP1) are targets for available drugs: fluvastatin and/or celecoxib. RESULTS: Quantification by RT-Q-PCR of the expression of these genes was performed in a series of 51 pilocytic astrocytomas and 10 glioblastomas: they were all significantly overexpressed in hypothalamo-chiasmatic pilocytic astrocytomas relative to cerebellar pilocytic astrocytomas, and CRK and ICAM1 were significantly overexpressed in pilocytic astrocytomas versus glioblastomas.We used two commercially available glioblastoma cell lines and three pilocytic astrocytoma explant cultures to investigate the effect of celecoxib/fluvastatin alone or in combination. Glioblastoma cell lines were sensitive to both drugs and a combination of 100 µM celecoxib and 240 µM fluvastatin was the most synergistic. This synergistic combination was used on the explant cultures and led to massive cell death of pilocytic astrocytoma cells.As a proof of concept, a patient with a refractory multifocal pilocytic astrocytoma was successfully treated with the fluvastatin/celecoxib combination used for 18 months. It was well tolerated and led to a partial tumor response. CONCLUSION: This study reports evidence for new targets and synergistic effect of celecoxib/fluvastatin combination in pilocytic astrocytoma. Because it is non-toxic, this new strategy offers hope for the treatment of patients with refractory pilocytic astrocytoma.