DZ binds to the vinca alkaloid site in a unique way, producing higher anti-tumor properties and safety. The electron microscopy and X-ray crystallography showed that DZ modifies the curvature between tubulin dimers, thus straightening protofilaments.
It enhances the rescue frequency, and, despite the limited effect on microtubules destabilization compared with vinorelbine, it is adequately sufficient to disrupt mitotic spindle formation. Vinorelbine was also effective in both xenograft models, but at higher doses and for a shorter term. The importance of DZ also lies in its increased safety margin more than fold vs 0.
A combination of DZ and gemcitabine was observed to be more efficacious than gemcitabine monotherapy, which is the first-line treatment of patients with PDAC. These results are indicative of DZ being a possible candidate for PDAC treatment and its potential to be used in a wide range of other applications MSAs mainly promote the polymerization of microtubules, making them unusually stable and increasing their quantities in the cell So far, only the taxane-site ligands were shown to have potent activity against PDAC.
The structure of paclitaxel was discovered in , but its microtubule-stabilizing characteristics were identified only 8 years later, in Generally, the process of microtubule polymerization requires GTP, but paclitaxel can promote tubulin polymerization without it.
Paclitaxel promotes microtubule polymerization at low concentration and temperature without significantly rising polymer levels of the microtubule 78 , Paclitaxel is one of the most effective microtubule-targeting anti-cancer drugs. Paclitaxel was approved by the FDA in and is stillconsidered to be one of the most critical supplements to chemotherapeutic regimens against various cancers, including PC At present, paclitaxel combined with albumin-based chemotherapy is used as the first line of advanced PC therapy.
Paclitaxel influences the dynamics and microtubule polymerization via binding to the taxane site, which leads to cell cycle arrest and cell death. Because paclitaxel dramatically decreases cell proliferation and mitotic rate of microtubules at low concentrations without significantly rising polymer levels, suppression of microtubule dynamics appears to be its most effective mechanism of mitotic arrest. Paclitaxel at high concentrations promotes the addition of tubulin dimers and disturbances ina dynamic balance of microtubules but acts the opposite at low concentrations Several approaches have been implemented to improve the solubility and pharmacology of paclitaxel, including albumin nanoparticles, liposomes, and emulsions Albumin-stabilized nanoparticle formulation of paclitaxel is also known as ABI , or nab-paclitaxel.
Researchers found enormous levels of fibrotic tissue in the tumor microenvironment. The visually impressive decrease in fibrotic tissue mass was noted in tumor tissues after administration of nab-paclitaxel compared with those treated withpaclitaxel. Nab-paclitaxel therapy decreased the amount of proliferating carcinoma cells to a greater extent than paclitaxel therapy as evidenced by a decreased amount of carcinoma cells expressing Ki Plasma and intratumor concentrations of paclitaxel following nab-paclitaxel or paclitaxel therapy were performed to investigate the potential mechanism of the therapeutic effectiveness of nab-paclitaxel over paclitaxel.
Nab-paclitaxel therapy was correlated with higher tumor stroma in the tumor microenvironment compared with paclitaxel-treated and untreated tumors. According to the results from both clinical and preclinical studies, the efficacy of nab-paclitaxel is superior to that of cremophor-based paclitaxelowing to many factors including a better pharmacokinetics behavior.
A higher intratumor paclitaxel concentration was achieved after nab-paclitaxel treatment that resulted in desmoplastic tumor stroma destruction and enhanced neoplastic cell death.
This may be another reason for the superiority of nab-paclitaxel over paclitaxel treatment in PDAC Apart from hindering cell division via interrupting the microtubule network, it can enhance transportation of paclitaxel to endothelial and tumor cells.
Nab-paclitaxel has many advantages compared with sb-paclitaxel. For instance, it produces significantly higher doses of paclitaxel in a shorter transfusion time 30 min vs 3 h for sb-paclitaxel , it can reach a higher peak concentration, enhance drug combination to tumors and endothelial cells more effectively. Another study showed the nab-paclitaxel has a higher neoplasm uptake thansb-paclitaxel after administration at equal doses.
Median overall survival was significantly longer in the nab-paclitaxel plus gemcitabine group 8. Other trials reported that the grade 3 neuropathy was correlated with nab-paclitaxel treatment in a majority of patients with advanced PC Addition of nab-paclitaxel or docetaxel at IC25 reduced IC50 of gemcitabine.
Furthermore, a PDAC xenograft model study showed that nab-paclitaxel is more efficacious and results in longer median survival than gemcitabine. Nab-paclitaxel plus gemcitabine therapy comprises standards of metastatic PC care, and this combination is suitable for PDAC patients with different characteristics and clinical presentations Secreted protein acidic and rich in cysteine SPARC has a crucial role in the transport of nab-paclitaxel to a tumor.
A research was conducted to examine the relationship between the prognosis of patients receiving nab-paclitaxel plus gemcitabine and SPARC expression As technologies advance, nab-paclitaxel undergoes additional investigations for PDAC therapy. The solvent-free albumin-paclitaxel nanoparticles are comparatively more favorable than solvent-based formulations of cre-paclitaxel in patients with advanced metastatic PC.
Stopping treatment with albumin-paclitaxel is associated with a lower risk of neutropenia, infusion hypersensitivity responses, and quicker alleviation of external neuropathy. Albumin-paclitaxel is currently regarded as an ideal regimen for patients with metastatic PDAC. Albumin-bound formulation reduces tumor stroma via synergy between albumin and SPARC, thereby affecting the tumor microenvironment. This mechanism promotes the gemcitabine-enhanced effect.
Several studies examined the efficacy and survival advantage of nab-paclitaxel alone and in combination with gemcitabine. They aimed to study treatment effects on tumor cell proliferation, tumor desmoplasia, and metastases to adjacent organs Nab-paclitaxel as an individual agent was not found to be significantly useful in decreasing primary tumor weight or increasing mouse survival rate compared with nab-paclitaxel or gemcitabine monotherapy. Finally, combined treatment of gemcitabine and nab-paclitaxel reduced metastatic tumor burden and elevated median survival rate of animals greater than any of the agents alone 87 , The synergy between nab-paclitaxel and gemcitabine in PDAC was assessed in two preclinical models: genetically engineered mice and primary patient-derived tumors.
Combined treatment of gemcitabine plus nab-paclitaxel reduced metastatic tumor burden and improved the overall survival rate of animals compared with monotherapy of any of the agents.
Moreover, there is no benefit of adding paclitaxel to gemcitabine treatment for regionally advanced and metastatic PDAC In , gemcitabine plus nab-paclitaxel was approved by the FDA as the first-line treatment for patients with metastatic PC.
Nab-paclitaxel plus gemcitabine could better improve tumor response and survival rates in metastatic PDAC than gemcitabine alone Treatment with nab-paclitaxel seemed to exhaust the desmoplastic stromal matrix and improve microvasculature in gemcitabine-resistant primary tumors.
Intratumoral gemcitabine concentration was 2. Related synergistic anti-tumor and pharmacologic responses were confirmed in a transgenic PDAC murine model. The results showed that paclitaxel elevated intratumoral accumulation of gemcitabine via inactivation of cytidine deaminase. Another study revealed that nab-paclitaxel plus gemcitabine therapy efficiently reduced the density of tumor-associated fibroblasts and produced disruptive changes in tumor stroma.
Preclinical trial results revealed the positive anti-tumor activity of nab-paclitaxel and its potential to alter desmoplastic stroma. The results of this research lead to regulatory approval of nab-paclitaxel plus gemcitabine therapy, which is now a standard regimen for metastatic PC Epothilones are a novel class of anti-microtubule agents derived from the soil bacterium Sorangium cellulose.
They bind to the taxane site and stabilize microtubule polymerization. Epothilones have the activity of promoting assembly and polymerization of microtubules. After binding to the microtubule, epothilones restructure the disordered M-loop site of lateral tubulin contacts within the microtubule and stabilize it.
Compared with paclitaxel, epothilones have several advantages. First, the activity of epothilones is 10 to 1, times higher than that of paclitaxel. Second, the water solubility is also higher.
Lastly, the structure is much simpler, making them easier to synthesize This derivative is named ixabepilone, previously known as BMS Ixabepilone is more efficient in inhibiting tumor growth than paclitaxel with five to six lower doses required in mice and rats. The treatment with this compound resulted in median survival of 7. Z arylarylaminopropenone 10 compounds enhance microtubule stability and induce cell apoptosis via caspase family Z 2-bromo-3,4,5-trimethoxyphenyl 3-hydroxymethoxyphenino -propenone 10ae promotes tubulin polymerization and induces apoptotic cell death in MIA-Paca2 and Panc-1 cell lines It induces apoptosis in 20 tumor cell lines with similar GI50 values, including drug-resistant tumor cell lines.
Such a broad spectrum of action is probably due to its inhibitory effect on critical stages of cancer cell division. The influence of 10ae on the phosphorylation of proteins serving as markers of SAC activation and mitotic arrest history H3, Bcl-2, and BubR1 was then studied. All three were phosphorylated 6 h after 10ae treatment, showing a similar increase in concentration level 2.
To conclude it all, 10ae has the most potent cytotoxic properties among the other derivatives of 10 compounds. MTAs alter the normal structure and function of microtubules that subsequently affects microtubule assembly and spindles formation Figure 3. Mitotic spindles lose the pulling power required to separate sister chromatids and cannot properly orientate them, which completely stops the process of cell division. The two-way separation of sister chromatids is regulated by SAC. To ensure that anaphase does not start when kinetochores are not attached or attached improperly, checkpoint proteins are recruited on kinetochores and form a mitotic checkpoint complex MCC.
Errors in the test point of the cell cycle of tumor cells may result in drug sensitivity differences due to changes in the structure or expression of test point kinase Figure 3 Mechanism of microtubule-targeting drugs in cancer therapy. Improper, incomplete or absent attachment at kinetochores maintain spindle assembly checkpoint SAC activity. Produced cytochrome c bind to apoptosis-protease activating factor 1 Apaf1 and result in the generation of the apoptosome.
Eventually, a caspase cascade is triggered leading to apoptosis. Tumor cells increase endothelial cell proliferation and vasopermeability and alter gene expression via vascular endothelial growth factor VEGF pathway. Ensuing angiogenesis facilitates tumor cell proliferation.
MTAs cut off tumor blood supply by destroying its vasculature. Increase in p53 concentration stimulates the production of p27 that inhibits cyclin-dependent kinase Cdks and thus prevents cell cycle transition at several checkpoints. P53 can also interact with some members of the Bcl-2 family and induce apoptosis via the aforementioned mechanism.
Paclitaxel can block the cell cycle via two following mechanisms: regulation of expression of cyclin B1 and cyclin-dependent kinase Cdk. Cell cycle arrest has long been known to trigger apoptosis. Apoptosis induction occurs via different pathways, such as phosphorylation of Bcl-2 and Bcl-xL, activation and upregulation of E2F1—all of which can instigate the release of cytochrome c Activation of mammalian target of rapamycin mTOR is also implicated with microtubules.
This independent mechanism represents a unique tool for inducing mitotic arrest Folkman et al. Destruction of tumor vasculature, starvation of tumor cells, and other strategies have been widely adopted in many types of cancer. Several cancers in mice were found to be inhibited after feeding with natural vascular inhibitors The application of MTAs becomes a new direction in the research of anti-tumor drugs.
Drugs, such as paclitaxel, vinblastine, and colchicine, have potential anti-angiogenic effects on PC. Vinblastine was shown to exhibit dose-dependent anti-angiogenic activity in a chick embryo chorioallantoic membrane model. A research confirmed that tumor blood vessels could be selectively destroyed within 6 h after administration of CA-4 in an alive rat model.
Notably, the effect on the intratumor blood vessels is stronger than on the extratumor ones. Some researchers are currently investigating the effects of MTAs on vascular destruction and conducting corresponding clinical trials. More than 10 types of tumor vasculature-targeting drugs are enrolled in clinical trials. Additionally, a prolonged mitotic arrest can lead to DNA damage, which activates a DNA damage response that is pdependent leading to subsequent apoptosis [ 9 , 10 ].
However, cancer cells often misregulate their cell cycle checkpoints and can have varied responses to antimitotics; not only between different types of cancers but also within the same type of cancer cells [ 5 ]. Therefore, cancer cells treated with antimitotics may bypass the SAC and undergo an aberrant division, which can later lead to apoptosis from any cell cycle phase [ 11 ].
Additionally, it is now well established that the cytotoxic effect of antimitotics is in part due to the disruption of interphase cytoskeletal microtubules [ 12 ]. Thus, the effect of antimitotics on cancer cells goes beyond their ability to inhibit cell division.
Although microtubule targeting agents are some of the most common chemotherapeutic agents used to treat a wide variety of cancers, they show important dose-limiting toxicities, including neutropenia and neurotoxicity, largely a consequence of disturbing microtubule dynamics in neurons [ 13 , 14 ]. Most of the microtubule targeting agents used clinically are large, natural difficult to synthesize , hydrophobic compounds with limited solubility.
Thus, there is a critical need to identify novel tubulin-targeting drugs with improved properties that can be used as anticancer agents. Here, we have discovered and characterized E styryl-5,6,7,8-tetrahydrobenzo [ 4 , 5 ] thieno[2,3-d]pyrimidin-4 3H -one Microtubin-1 and its analogues Microtubins ; a novel class of drug-like microtubule targeting agents that inhibit cancer cell proliferation.
The Microtubins disrupt microtubule polymerization, arrest cells in mitosis, activate the spindle assembly checkpoint and trigger an apoptotic cell death. Importantly, the Microtubins do not compete with vinblastine for the known vinca binding site or colchicine for the colchicine binding site and inhibit tubulin polymerization through a different mechanism.
Microtubin structure-activity relationship SAR studies indicated that modification of the Microtubin phenyl ring was critical to modulating its ability to inhibit microtubule polymerization, formation of the mitotic spindle, and cell division. Additionally, the Microtubins were not only active against a cervical adenocarcinoma cell line, but also patient derived glioblastoma cells and multi-drug resistant small cell lung carcinoma cells.
Thus, the Microtubins represent a novel class of compounds that could be developed for therapeutic use in the treatment of cancer.
Briefly, Human cervical adenocarcinoma HeLa cells were treated with DMSO or one of the 79, compounds in the library for twenty hours and their cell cycle profile was analyzed using the DNA-selective stain Vybrant DyeCycle Green; a cell membrane permeant dye that binds to DNA and emits a fluorescent signal that is proportional to DNA mass when excited at nm with a cytometer [ 17 ]. This approach yielded E styryl-5,6,7,8-tetrahydrobenzo [ 4 , 5 ] thieno[2,3-d]pyrimidin-4 3H -one Microtubin-1 Figure 1A.
Microtubin-1 is a small Table 1: Predicted chemical properties of Microtubins and other microtubule targeting agents. Figure 1: Identification of Microtubin-1, a novel cell division inhibitor. C , HeLa cells were treated with increasing concentrations of colchicine and Microtubin-1 and the drug response dose curves were used to measure the mitotic arrest IC 50s Vybrant DyeCycle Green assay and the cell viability IC 50s CellTiter-Glo assay for each treatment.
Error bars indicate standard deviations from 3 independent triplicate experiments. To determine whether Microtubintreated cells were arresting in mitosis or G2 phase, we performed immunofluorescence microscopy on cells that had been treated with colchicine or Microtubin-1 for 20 hours.
This analysis indicated that colchicine and Microtubintreated cells arrested in mitosis positive for p-H3 with condensed chromosomes and depolymerized microtubules [ 21 , 22 ] Figure 1B. To determine if Microtubin-1 arrested mitotic cells were dying, we utilized the same drug titration series to treat cells for 72 hours and the cell viability was measured using the CellTiter-Glo luminescent cell viability assay, which measures total ATP levels indicative of metabolically active cells using a luminometer at nm wavelength.
The cell viability IC 50 was then quantified. Next, we asked if the Microtubin-1 induced cell death was through caspase dependent apoptosis. Together these results indicated that Microtubin-1 was inhibiting microtubule polymerization, which arrested cells in mitosis and activated an apoptotic cell death to decrease the viability of cervical adenocarcinoma cells.
The mechanism of action for microtubule depolymerizing agents can be classified on the basis of where they bind to within tubulin, which include the vinca site bound by large natural compounds like the vinca alkaloids vincristine and vinblastine and the colchicine site bound by small compounds like colchicine and podophyllotoxin [ 23 , 24 ].
Thus, we used a mass spectrometry-based competition assay to determine if Microtubin-1 was binding to either of these two sites or to a novel site [ 25 , 26 ]. First, we analyzed whether Microtubin-1 was able to compete the vinblastine-tubulin interaction compared to vincristine, which binds to the vinca site.
This analysis showed that Microtubin-1 was not able to compete the vinblastine-tubulin interaction similar to a negative control compound 34 C34 , whereas vincristine VCR was able to compete this interaction Figure 2A. Similarly, we analyzed the ability of Microtubin-1 to compete the colchicine-tubulin interaction compared to podophyllotoxin, which binds the colchicine site.
Interestingly, Microtubin-1 was also not able to compete this interaction similar to the negative control vincristine VCR , whereas podophyllotoxin podo was able to compete this interaction Figure 2B.
These results indicated that Microtubin-1 was not binding to the vinca or colchicine sites and was likely targeting a novel site. Figure 2: Microtubin-1 does not compete for binding to the vinca-binding site or the colchicine-binding site. A-B , mass spectrometry-based competitive binding assays to test the binding of Microtubin-1 Mtbin-1 to the vinca A and colchicine B site. A, Microtubin-1 does not compete with vinblastine for binding to the vinca site compared to the positive control vincristine VCR.
C34 is the negative control compound B, Microtubin-1 does not compete with colchicine for the colchicine site compared to the positive control podophyllotoxin Podo. To improve the antiproliferative activity of Microtubin-1 without knowledge of its binding site and to understand the chemical properties that influence Microtubin-1 activity, we took two complementary ligand-based approaches Figure 3.
This approach yielded compounds with varied functional group additions to the core scaffold of Microtubin-1 Figure 3 and Supplementary Table 1. As an alternative approach, we evaluated the synthetic routes based on the core structure of Microtubin-1 for ease of chemical modification and subsequently designed 38 phenyl ring derivatives based on the Topliss scheme for aromatic ring optimization [ 28 ] Figure 3 and Supplementary Table 2.
We then selected and acquired or synthesized Supplementary Figure 1 13 analogues from each group a total of 26 analogues with diverse functional modifications for further testing Figure 3 , Table 2 and Supplementary Table 3.
Figure 3: Microtubin-1 optimization. Additionally, the Topliss scheme for aromatic ring optimization was used to identify 38 Microtubin-1 phenyl ring derivatives. The top 13 drug-like analogues and 13 drug-like phenyl derivatives of Microtubin-1 were selected for testing in HeLa cell culture structure activity relationship studies.
To understand the chemical parameters that were important for the activity of Microtubin-1 and to determine if Microtubin-1 analogues were more potent, we evaluated the 26 Microtubin-1 analogues in cell culture-based structure-activity relationship SAR studies.
The cell viability IC 50 was then quantified for each compound. The Microtubin SAR indicated that functionalization of the phenyl ring could improve Microtubin activity while substitution at other positions of the core scaffold, including the addition of a nitrile group at the R2 position, the addition of a 3-methyl group at the R3 position or ring expansion of the tricyclic core inactivated the compounds Table 2. The preferred aromatic ring substitutions included fluorination at the 2, 4, and 5 positions, while methoxy or sulfur methyl substitution at the 3 or 4 positions also led to an observed cytotoxicity Table 2.
The SAR also indicated that the position of the substituents was critical for the activity of these compounds. For example, addition of a 3-methoxyl group to Microtubin activated the compound Microtubin-7 , while further removal of the 4-ethoxy increased potency by 6 fold Microtubin-5 Table 2.
Among the active compounds including Microtubin-5, Microtubin-7 and Microtubin-9 the methoxy and its bioisostere sulfur methyl group primarily substituted the 3 and 4 positions of the phenyl ring but not at other positions Table 2. To further validate that Microtubin-1 and its analogues were targeting microtubules, we performed in vitro microtubule polymerization reactions with the most potent compounds Microtubin using an in vitro microtubule polymerization assay [ 26 ].
Microtubule polymerization was monitored by endpoint and kinetic measurements. For endpoint measurements, polymerization reactions were subjected to centrifugal sedimentation and the supernatant and pellet fractions were resolved by SDS-PAGE and gels were stained with Coomassie blue to visualize the quantity of polymerized microtubules in the pellet fraction versus non-polymerized tubulin in the supernatant Figure 4A.
For kinetic measurements, microtubule polymerization was monitored by reading the fluorescence at nm due to the incorporation of a fluorescent reporter into microtubules as polymerization occurred every minute using a Tecan M microplate reader Figure 4B.
Both endpoint and kinetic measurements indicated that in vitro Microtubin-1, Microtubin-2, and Microtubin-3 were inhibitors of microtubule polymerization similar to colchicine Figure 4A and 4B.
Next, we analyzed microtubule stability in HeLa cells treated with increasing concentrations of each of these three compounds. This analysis showed that, similar to colchicine, the microtubules of Microtubin-1, Microtubin-2, and Microtubintreated cells became destabilized in a drug dose-dependent manner Figure 4C.
Figure 4: The Microtubins inhibit microtubule polymerization in vitro and in cells. A, reaction products were subjected to centrifugal sedimentation and the supernatant Sup and pellet Pel fractions were resolved by SDS-PAGE and tubulin polymerization was visualized with Coomassie blue staining. B, microtubule polymerization was monitored over time every minute by measuring the absorbance at nm.
Graph displays fluorescence signal in arbitrary fluorescence units on the y-axis over time in minutes on the x-axis for the indicated drug treatments. C , immunofluorescence microscopy of cells treated with DMSO or increasing concentrations of taxol, colchicine, Microtubin-1, Microtubin-2, or Microtubin-3 for 20 hours. Colchicine treatment in man inhibits the adhesive activity of neutrophils tested ex vivo.
From Fordham et al. A common feature of these diseases, and to some extent also of normal neuronal aging, is a reduction in microtubule density in axons. Axonal microtubules provide the tracks for axonal transport, and probably also play important roles in signaling pathways. Stabilizing axonal microtubules with drugs might provide symptomatic relief in neurodegenerative disease, and perhaps even inhibit disease progression.
Consistent with this hypothesis, treatment of mice genetically engineered to model neurodegenerative diseases with microtubule-stabilizing drugs that enter the CNS caused improvement in neuropathology and behavioral symptoms Brunden et al.
Stabilizing microtubules was also shown to promote repair of spinal cord injury in animal models Ruschel et al. This, stabilizing microtubules in the brain and spinal cord might have multiple therapeutic benefits. A major challenge for translating this hypothesis to man is the potent anti-mitotic toxicity of current microtubule stabilizing drugs that penetrate the CNS, such as epothilones.
We will pursue several strategies to try and overcome this problem, including development of novel chemical scaffolds that stabilize microtubules and exhibit improved ratios of neuro-protective to antimitotic activity in the hope of better treatments for these devastating diseases.
Figure 4. Epothilone treatment induces microtubule polymerization and neurite outgrowth in a cell culture model of spinal cord damage. From Ruschel et al. Brunden, K. Microtubule-stabilizing agents as potential therapeutics for neurodegenerative disease. Chittajallu, D. T cells are also negatively controlled by immune checkpoint proteins, classes of molecules and signals that restrain T-cell proliferation, survival, and activation Although cancer cells can express tumor-specific neoantigens, thus being susceptible to be targeted by the immune system, cancer cells often express on their surface immune checkpoint molecules that suppress activation of T cells that could grant tumor immune surveillance.
Based on these observations, immunotherapies targeting these molecules have been developed and are emerging as a major breakthrough in cancer treatment 36 , The exact mechanism by which anti-CTLA-4 antibodies induce an antitumor response is still imprecisely known, although preclinical evidence suggests that CTLA-4 blockade supports the activation and proliferation of a higher number of effector T cells and reduces TReg cell-mediated suppression of effector T-cell response 39 , Indeed, after successful clinical trials, the anti-CTLA-4 monoclonal antibody ipilimumab was first approved for the treatment of advanced or unresectable melanoma 41 , PD-1, a cell surface receptor, is expressed on regulatory and cytotoxic activated T cells in peripheral tissues while PD-L1 is mainly expressed on APC.
Physiologically, binding of PD-L1 to its receptor results in T-cell inactivation. PD-1 is highly expressed on many tumor-infiltrating lymphocytes and cancer cells often overexpress PD-L1, thus escaping immune surveillance Indeed, antibodies blocking the binding of PD-L1 to its receptor, such as nivolumab and pembrolizumab, enhance immunity against a wide variety of cancers 37 , FDA rapidly approved these drugs for the treatment of melanoma, urothelial cancer, renal cell carcinoma, non-small-cell lung cancer NSCLC , Hodgkin lymphoma, and squamous cell carcinoma of head and neck.
A possible strategy to improve ICI-based therapy in cancer patients is to combine it with radiation or traditional, DNA-damaging, antiblastic therapies. It was soon hypothesized that the combination of DNA-damaging radiations or drugs with ICIs could be highly beneficial to cancer patients. Increasing evidence suggests that the antitumor activity of DNA-damaging treatments is mediated not only through cytotoxic effects, but also because they stimulate immune surveillance by affecting both cancer and immune cells For example, some DNA-alkylating agents, like cyclophosphamide and carboplatin, or antimetabolites, like pemetrexed, both increase the expression of MHC class I molecules on cancer cells and subvert the immunosuppressive functions of TReg cells 50 , Cells with double-stranded DNA breaks that progress through mitosis accumulate micronuclei 53 , Remarkably, using a well-described B16 syngenic mouse model of melanoma, it has been shown that irradiation of one tumor along with immune checkpoint blockade results in a T-cell-dependent growth delay of a contralateral unirradiated tumor The results obtained in the melanoma mouse model, therefore, suggest that the immune checkpoint manipulation could indeed enhance the response to DNA-damaging, radio- and chemotherapies.
In any case, limitations of combining DNA-damaging radiations or drugs with ICIs appear essentially due to lack of ICI effects because of damage to immune cells by the DNA-damaging agents or, conversely, to adverse, toxic, effects of hyperactivation of inflammatory, and immunological reaction towards normal tissues 57 , The fact that most of the spindle-targeting drugs are not so efficacious in cancer treatment has reinforced the idea that MTAs also act independently of their ability to delay mitosis completion How MTAs might kill cells besides their ability to induce mitotic cell death, is, however, mechanistically unclear and object of great debate 8 , 13 , 14 , 59 , 60 , Thus, as recently suggested, taxane-based therapy may also work because it elicits the antitumor intervention of the immune system on cells escaping mitotic death 8 , Thus, taxane-based therapies could benefit by the combination with ICIs.
Several clinical trials have also been designed now to explore the effect of a combinatorial therapy with taxanes and ICIs. The majority of these clinical trials are still ongoing and their preliminary but very promising results are still to be definitively proven.
It is a fact, however, that upon successful completion of two such trials, pembrolizumab and atezolizumab, another anti-PD-L1 monoclonal antibody, have been approved in combination with paclitaxel or its albumin-stabilized nanoparticle formulation nab-paclitaxel for the first-line treatment of metastatic squamous NSCLC 66 , Moreover, atezolizumab in combination with the sole nab-paclitaxel has also been approved for the treatment of women with unresectable triple-negative breast cancer The efficacy of the combination of taxanes and ICIs in cancer therapy may be explained by a simple additive effect of the two classes of drugs.
However, as already discussed, the complex and not yet completely investigated immunomodulatory activity of MTAs on tumor-infiltrating immune cells might at least in part explain the success of the MTAs and ICIs combination Death after prolonged mitosis or following slippage is certainly a way MTAs kill cancer cells. However, in the case of paclitaxel, recent correlations between clinical therapeutic success for breast cancer patients and the type of mitotic aberrations induced by this drug in their breast cancer cells have indicated that the therapeutic benefit correlates with alterations in chromosome segregation rather than with prolongation of the duration of mitosis Indeed, while at relatively high doses paclitaxel induces mitotic delay, at much lower concentrations it does not significantly delay mitosis duration but perturbs its normal execution inducing a significant degree of chromosome missegregation and formation of micronuclei in daughter cells Fig.
When single or small groups of chromosomes do not segregate with the mass of other chromosomes, they become wrapped up in nuclear membranes and remain separate from the primary nucleus 8.
Thus, micronuclei spontaneously and frequently lose nuclear envelope integrity, generating further DNA damage Cancer cells may express tumor-specific neoantigens and when treated with low doses of taxanes may induce micronucleation-dependent activation of antigen presenting cell APC. These observations suggest not only that a major reason for the therapeutic success of taxanes, and perhaps of other classes of MTAs, relies on their ability to promote antitumor immune surveillance but also that low doses of the drugs may be sufficient to achieve this goal.
However, immune checkpoint may oppose to antitumor immune surveillance stimulated by taxanes Fig. Based on these considerations, we would like to propose the use of low doses of taxanes in combination with ICIs as a strategy of wide applicability, high tolerability, and efficacy in cancer treatment Fig.
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