Microtubule and tubulin binding and regulation of microtubule dynamics by the antibody drug conjugate (ADC) payload, monomethyl auristatin E (MMAE): Mechanistic insights into MMAE ADC peripheral neuropathy.

Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy.

Plinabulin, an inhibitor of tubulin polymerization, targets KRAS signaling through disruption of endosomal recycling.

Constitutive activation of Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most common oncogenic event in certain types of human cancer and is associated with poor patient survival. Small molecule signaling inhibitors have improved the clinical outcomes of patients with various cancer types but attempts to target KRAS have been unsuccessful. Plinabulin represents a novel class of agents that inhibit tubulin polymerization with a favorable safety profile in clinical trials. In the present study, the potency of plinabulin to inhibit tubulin polymerization and growth of KRAS-driven cancer cells was characterized. In vivo efficacy of plinabulin was tested in two different mouse models; one being the RCAS/t-va gene transfer system and the other being a xenograft model. In vitro cell culture tubulin polymerization assays were used to complement the mouse models. There was improved survival in a KRAS-driven mouse gene transfer glioma model, but lack of benefit in a similar model, without constitutively active KRAS, which supports the notion of a KRAS-specific effect. This survival benefit was mediated, at least in part, by the ability of plinabulin to inhibit tubulin polymerization and disrupt endosomal recycling. It was proposed a mechanism of compromised endosomal recycling of displaced KRAS through targeting microtubules that yields inhibition of protein kinase B, but not extracellular signal regulated kinase (ERK) signaling, therefore lending rationale to combination treatments of tubulin- and ERK-targeting agents in KRAS-driven cancer.

ßIII-tubulin enhances efficacy of cabazitaxel as compared with docetaxel.

Cabazitaxel is a novel taxane approved for treatment of metastatic hormone-refractory prostate cancer in patients pretreated with docetaxel. Cabazitaxel, docetaxel, and paclitaxel bind specifically to tubulin in microtubules, disrupting functions essential to tumor growth. High levels of ßIII-tubulin isotype expression are associated with tumor aggressivity and drug resistance. To understand cabazitaxel's increased efficacy, we examined binding of radio-labeled cabazitaxel and docetaxel to microtubules and the drugs' suppression of microtubule dynamic instability in vitro in microtubules assembled from purified bovine brain tubulin containing or devoid of ßIII-tubulin. We found that cabazitaxel suppresses microtubule dynamic instability significantly more potently in the presence of ßIII-tubulin than in its absence. In contrast, docetaxel showed no ßIII-tubulin-enhanced microtubule stabilization. We also asked if the selective potency of cabazitaxel on ßIII-tubulin-containing purified microtubules in vitro extends to cabazitaxel's effects in human tumor cells. Using MCF7 human breast adenocarcinoma cells, we found that cabazitaxel also suppressed microtubule shortening rates, shortening lengths, and dynamicity significantly more strongly in cells with normal levels of ßIII-tubulin than after 50% reduction of ßIII-tubulin expression by siRNA knockdown. Cabazitaxel also more strongly induced mitotic arrest in MCF7 cells with normal ßIII-tubulin levels than after ßIII-tubulin reduction. In contrast, docetaxel had little or no ßIII-tubulin-dependent selective effect on microtubule dynamics or mitotic arrest. The selective potency of cabazitaxel on purified ßIII-tubulin-containing microtubules and in cells expressing ßIII-tubulin suggests that cabazitaxel may be unusual among microtubule-targeted drugs in its superior anti-tumor efficacy in tumors overexpressing ßIII-tubulin.

Microtubule-Targeting Agents Eribulin and Paclitaxel Differentially Affect Neuronal Cell Bodies in Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of anticancer treatment with microtubule-targeted agents (MTAs). The frequency of severe CIPN, which can be dose limiting and even life threatening, varies widely among different MTAs. For example, paclitaxel induces a higher frequency of severe CIPN than does eribulin. Different MTAs also possess distinct mechanisms of microtubule-targeted action. Recently, we demonstrated that paclitaxel and eribulin differentially affect sciatic nerve axons, with paclitaxel inducing more pronounced neurodegenerative effects and eribulin inducing greater microtubule stabilizing biochemical effects. Here, we complement and extend these axonal studies by assessing the effects of paclitaxel and eribulin in the cell bodies of sciatic nerve axons, housed in the dorsal root ganglia (DRG). Importantly, the microtubule network in cell bodies is known to be significantly more dynamic than in axons. Paclitaxel induced activating transcription factor 3 expression, a marker of neuronal stress/injury. Paclitaxel also increased expression levels of acetylated tubulin and end binding protein 1, markers of microtubule stability and growth, respectively. These effects are hypothesized to be detrimental to the dynamic microtubule network within the cell bodies. In contrast, eribulin had no significant effect on any of these parameters in the cell bodies. Taken together, DRG cell bodies and their axons, two distinct neuronal cell compartments, contain functionally distinct microtubule networks that exhibit unique biochemical responses to different MTA treatments. We hypothesize that these distinct mechanistic actions may underlie the variability seen in the initiation, progression, persistence, and recovery from CIPN.

Structural Basis for Induction of Peripheral Neuropathy by Microtubule-Targeting Cancer Drugs.

Peripheral neuropathy is a serious, dose-limiting side effect of cancer treatment with microtubule-targeting drugs. Symptoms present in a "stocking-glove" distribution, with longest nerves affected most acutely, suggesting a length-dependent component to the toxicity. Axonal transport of ATP-producing mitochondria along neuronal microtubules from cell body to synapse is crucial to neuronal function. We compared the effects of the drugs paclitaxel and ixabepilone that bind along the lengths of microtubules and the drugs eribulin and vincristine that bind at microtubule ends, on mitochondrial trafficking in cultured human neuronal SK-N-SH cells and on axonal transport in mouse sciatic nerves. Antiproliferative concentrations of paclitaxel and ixabepilone significantly inhibited the anterograde transport velocity of mitochondria in neuronal cells, whereas eribulin and vincristine inhibited transport only at significantly higher concentrations. Confirming these observations, anterogradely transported amyloid precursor protein accumulated in ligated sciatic nerves of control and eribulin-treated mice, but not in paclitaxel-treated mice, indicating that paclitaxel inhibited anterograde axonal transport, whereas eribulin did not. Electron microscopy of sciatic nerves of paclitaxel-treated mice showed reduced organelle accumulation proximal to the ligation consistent with inhibition of anterograde (kinesin based) transport by paclitaxel. In contrast, none of the drugs significantly affected retrograde (dynein based) transport in neuronal cells or mouse nerves. Collectively, these results suggest that paclitaxel and ixabepilone, which bind along the lengths and stabilize microtubules, inhibit kinesin-based axonal transport, but not dynein-based transport, whereas the microtubule-destabilizing drugs, eribulin and vincristine, which bind preferentially to microtubule ends, have significantly less effect on all microtubule-based axonal transport. Cancer Res; 76(17); 5115-23. ©2016 AACR.

Effects of Paclitaxel and Eribulin in Mouse Sciatic Nerve: A Microtubule-Based Rationale for the Differential Induction of Chemotherapy-Induced Peripheral Neuropathy.

Microtubule targeting agents (MTAs) often lead to treatment limiting and life threatening side effects, including chemotherapy-induced peripheral neuropathy (CIPN). The frequency of severe CIPN varies among different MTAs. Since the microtubule binding interactions and mechanisms of action also vary among MTAs, we hypothesized that these distinct mechanisms may underlie the variability in frequency of severe CIPN. Using a two-week, maximum tolerated dose model, we morphologically and biochemically analyzed sciatic nerves from mice treated with either paclitaxel or eribulin. These drugs differ in their manner of microtubule binding and mechanisms of action and reports indicate paclitaxel also induces a higher frequency of severe CIPN than does eribulin. Morphologically, paclitaxel increased the frequency of observed signs of axon degeneration more significantly than did eribulin. Alternatively, eribulin but not paclitaxel induced occasional myelin "halo" structures. Biochemically, paclitaxel, and eribulin both induced α-tubulin expression (~1.9- and ~2.5-fold, respectively) and tubulin acetylation, a marker for microtubule stability, (~5- and ~11.7-fold, respectively). Eribulin but not paclitaxel-induced EB1 expression ~2.2-fold while paclitaxel but not eribulin mildly suppressed EB3 expression. Both EB proteins are associated with microtubule growth. Eribulin's combination of relatively mild deleterious morphological effects coupled with more potent biochemical changes promoting microtubule stability and growth in mice correlate with lower frequencies of severe CIPN in humans. We suggest that these eribulin-induced effects create a relatively stable microtubule network that compensates, in part, for the toxic anti-cancer effects of the drug, leading to fewer reported incidences of CIPN than for paclitaxel.

Effects of eribulin on microtubule binding and dynamic instability are strengthened in the absence of the ßIII tubulin isotype.

Eribulin mesylate (Halaven) is a microtubule-targeted anticancer drug used to treat patients with metastatic breast cancer who have previously received a taxane and an anthracycline. It binds at the plus ends of microtubules and has been shown to suppress plus end growth selectively. Because the class III ß tubulin isotype is associated with resistance to microtubule targeting drugs, we sought to determine how ßIII tubulin might mechanistically influence the effects of eribulin on microtubules. We found that while [(3)H]eribulin bound to bovine brain soluble tubulin depleted of ßIII tubulin in a manner similar to that of unfractionated tubulin, it bound to plus ends of microtubules that were depleted of ßIII-depleted tubulin with a maximal stoichiometry (20 ± 3 molecules per microtubule) higher than that of unfractionated microtubules (9 ± 2 molecules per microtubule). In addition, eribulin suppressed the dynamic instability behavior of ßIII-depleted microtubules more strongly than and in a manner different from that of microtubules containing ßIII tubulin. Specifically, with ßIII tubulin present in the microtubules, 100 nM eribulin suppressed the growth rate by 32% and marginally reduced the catastrophe frequency (by 17%) but did not modulate the rescue frequency. However, in the absence of ßIII tubulin, eribulin not only reduced the growth rate but also strongly reduced the shortening rate (by 43%) and the catastrophe and the rescue frequencies (by 49 and 32%, respectively). Thus, when present in microtubules, ßIII tubulin substantially weakens the effects of eribulin.

Mechanism of action of ixabepilone and its interactions with the ßIII-tubulin isotype.

Ixabepilone (Ixempra, BMS-247550), a semisynthetic analog of epothilone B, is a microtubule-targeted drug in clinical use for treatment of metastatic or locally advanced breast cancer. Ixabepilone's binding and mechanism of action on microtubules and their dynamics, as well as its interactions with isotypically altered microtubules, both in vitro and in tumor cells, have not been described. Microtubules are dynamic polymers of the protein tubulin that function in mitosis, intracellular transport, cell proliferation, and migration. They continually undergo dynamic instability, periods of slow growth and rapid shortening that are crucial to these cell functions. We determined ixabepilone's microtubule binding and polymerization effects in vitro and also determined its effects on inhibition of dynamic instability in vitro and in cells, both with and without removal of the ßIII isotype of tubulin. The ßIII isotype of tubulin is associated with drug resistance and tumor aggressivity. We found that removal (in vitro) and knockdown (in cells) of ßIII-tubulin led to increased inhibition of microtubule dynamic instability by ixabepilone. Depletion of ßIII-tubulin from MCF7 human breast cancer cells also induced increased mitotic arrest by ixabepilone. Thus, ßIII-tubulin expression suppresses the antitumor effects of ixabepilone, indicating that increased ßIII-tubulin may be an important contributor to the development of resistance to ixabepilone.

Antiproliferative mechanism of action of the novel taxane cabazitaxel as compared with the parent compound docetaxel in MCF7 breast cancer cells.

Cabazitaxel, a novel chemotherapeutic taxane, is effective against docetaxel-resistant cells and tumors. It is approved for treatment of metastatic hormone-refractory prostate cancer in patients pretreated with docetaxel. Objective responses have been observed in many other cancers, including pretreated metastatic breast cancer. Cabazitaxel and docetaxel share a high degree of structural similarity. The basis for cabazitaxel's efficacy is unclear, and its mechanism has not been described. We compared the effects of cabazitaxel and docetaxel on MCF7 human breast cancer cells expressing fluorescent tubulin. Both drugs inhibited cell proliferation (IC50s, cabazitaxel, 0.4 ± 0.1 nmol/L, docetaxel, 2.5 ± 0.5 nmol/L) and arrested cells in metaphase by inducing mitotic spindle abnormalities. Drug concentrations required for half-maximal mitotic arrest at 24 hours were similar (1.9 nmol/L cabazitaxel and 2.2 nmol/L docetaxel). Cabazitaxel suppressed microtubule dynamic instability significantly more potently than docetaxel. In particular, cabazitaxel (2 nmol/L) suppressed the microtubule shortening rate by 59% (compared with 49% for 2 nmol/L docetaxel), the growing rate by 33% (vs. 19%), and overall dynamicity by 83% (vs. 64%). Cabazitaxel was taken up into cells significantly faster than docetaxel, attaining an intracellular concentration of 25 µmol/L within 1 hour, compared with 10 hours for docetaxel. Importantly, after washing, the intracellular cabazitaxel concentration remained high, whereas the docetaxel concentration was significantly reduced. The data indicate that the potency of cabazitaxel in docetaxel-resistant tumors is due to stronger suppression of microtubule dynamics, faster drug uptake, and better intracellular retention than occurs with docetaxel.

Erucin, the major isothiocyanate in arugula (Eruca sativa), inhibits proliferation of MCF7 tumor cells by suppressing microtubule dynamics.

Consumption of cruciferous vegetables is associated with reduced risk of various types of cancer. Isothiocyanates including sulforaphane and erucin are believed to be responsible for this activity. Erucin [1-isothiocyanato-4-(methylthio)butane], which is metabolically and structurally related to sulforaphane, is present in large quantities in arugula (Eruca sativa, Mill.), kohlrabi and Chinese cabbage. However, its cancer preventive mechanisms remain poorly understood. We found that erucin inhibits proliferation of MCF7 breast cancer cells (IC50 = 28 µM) in parallel with cell cycle arrest at mitosis (IC50 = 13 µM) and apoptosis, by a mechanism consistent with impairment of microtubule dynamics. Concentrations of 5-15 µM erucin suppressed the dynamic instability of microtubules during interphase in the cells. Most dynamic instability parameters were inhibited, including the rates and extents of growing and shortening, the switching frequencies between growing and shortening, and the overall dynamicity. Much higher erucin concentrations were required to reduce the microtubule polymer mass. In addition, erucin suppressed dynamic instability of microtubules reassembled from purified tubulin in similar fashion. The effects of erucin on microtubule dynamics, like those of sulforaphane, are similar qualitatively to those of much more powerful clinically-used microtubule-targeting anticancer drugs, including taxanes and the vinca alkaloids. The results suggest that suppression of microtubule dynamics by erucin and the resulting impairment of critically important microtubule-dependent cell functions such as mitosis, cell migration and microtubule-based transport may be important in its cancer preventive activities.

Mechanisms of inhibition of endothelial cell migration by taxanes.

Several antiangiogenic mechanisms have been proposed for the widely-used cancer chemotherapeutic drugs taxotere (docetaxel) and taxol (paclitaxel), but none has been definitively identified. We analyzed their effects at a range of concentrations on migration and mitosis of human umbilical vein endothelial cells (HUVECs) on microtubule and focal adhesion morphology and microtubule dynamic instability during migration. Both taxanes inhibited migration by inhibiting both maintenance of the forward direction of the cell and by slowing migration over the entire contorted path length. At low (but not all) taxane concentrations that inhibit HUVEC migration, the shortening rates and shortening lengths of microtubules at the leading edge were strongly inhibited; peripheral microtubules were reduced in number and fewer targeted focal adhesions; focal adhesions doubled in length and became ring-shaped, elongate, and reduced in number; and an increase in stabilized microtubules occurred centrally. HUVEC migration was 13-19-fold more sensitive to taxanes than was mitosis confirming that taxanes exhibit significant effects in addition to mitotic arrest that may contribute to their overall antitumor effects. No relationship was detected between centrosome position and migration characteristics. The data suggest that taxanes inhibit migration, at least in part, by inhibiting the dynamic instability of microtubules that possibly interact with guanine nucleotide exchange factors and thus with the focal adhesion-associated G-proteins that then lead to disruption of the regulated formation and turnover of focal adhesions. A mechanism is presented by which subcytotoxic concentrations of taxanes might stabilize dynamic instability of a few microtubules and thereby inhibit migration and angiogenesis.

Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: implications for chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious, painful and dose-limiting side effect of cancer drugs that target microtubules. The mechanisms underlying the neuronal damage are unknown, but may include disruption of fast axonal transport, an essential microtubule-based process that moves cellular components over long distances between neuronal cell bodies and nerve terminals. This idea is supported by the "dying back" pattern of degeneration observed in CIPN, and by the selective vulnerability of sensory neurons bearing the longest axonal projections. In this study, we test the hypothesis that microtubule-targeting drugs disrupt fast axonal transport using vesicle motility assays in isolated squid axoplasm and a cell-free microtubule gliding assay with defined components. We compare four clinically-used drugs, eribulin, vincristine, paclitaxel and ixabepilone. Of these, eribulin is associated with a relatively low incidence of severe neuropathy, while vincristine has a relatively high incidence. In vesicle motility assays, we found that all four drugs inhibited anterograde (conventional kinesin-dependent) fast axonal transport, with the potency being vincristine=ixabepilone>paclitaxel=eribulin. Interestingly, eribulin and paclitaxel did not inhibit retrograde (cytoplasmic dynein-dependent) fast axonal transport, in contrast to vincristine and ixabepilone. Similarly, vincristine and ixabepilone both exerted significant inhibitory effects in an in vitro microtubule gliding assay consisting of recombinant kinesin (kinesin-1) and microtubules composed of purified bovine brain tubulin, whereas paclitaxel and eribulin had negligible effects. Our results suggest that (i) inhibition of microtubule-based fast axonal transport may be a significant contributor to neurotoxicity induced by microtubule-targeting drugs, and (ii) that individual microtubule-targeting drugs affect fast axonal transport through different mechanisms.

Modeling the effects of drug binding on the dynamic instability of microtubules.

We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady-state microtubules assembled from MAP-free tubulin in the possible presence of a microtubule-associated drug. As an example of the latter, we both experimentally and theoretically study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. Our model predicts that among the drugs that act locally at the microtubule tip, primary inhibition of the loss of GDP tubulin results in stronger damping of microtubule dynamics than inhibition of GTP tubulin addition. On the other hand, drugs whose action occurs in the interior of the microtubule need to be present in much higher concentrations to have visible effects.

Combinatorial Tau pseudophosphorylation: markedly different regulatory effects on microtubule assembly and dynamic instability than the sum of the individual parts.

Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr(231), Ser(262), Ser(396), and Ser(404)) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.

Characterization and detection of cellular and proteomic alterations in stable stathmin-overexpressing, taxol-resistant BT549 breast cancer cells using offgel IEF/PAGE difference gel electrophoresis.

Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.

Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules.

Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ≥2 µmol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar K(D) values (0.86 ± 0.2 and 0.93 ± 0.2 µmol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (K(D), 0.1 ± 0.05 µmol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule.

Maytansinoid-antibody conjugates induce mitotic arrest by suppressing microtubule dynamic instability.

Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule dynamic instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule dynamic instability.

Microtubule-binding agents: a dynamic field of cancer therapeutics.

Microtubules are dynamic filamentous cytoskeletal proteins composed of tubulin and are an important therapeutic target in tumour cells. Agents that bind to microtubules have been part of the pharmacopoeia of anticancer therapy for decades and until the advent of targeted therapy, microtubules were the only alternative to DNA as a therapeutic target in cancer. The screening of a range of botanical species and marine organisms has yielded promising new antitubulin agents with novel properties. In the current search for novel microtubule-binding agents, enhanced tumour specificity, reduced neurotoxicity and insensitivity to chemoresistance mechanisms are the three main objectives.

Determination of microtubule dynamic instability in living cells.

The precise regulation of microtubules and their dynamics is critical for cell cycle progression, cell signaling, intracellular transport, cell polarization, and organismal development. For example, mitosis, cell migration, and axonal outgrowth all involve rapid and dramatic changes in microtubule organization and dynamics. Microtubule-associated proteins (MAPs) such as MAP2 and tau (Bunker et al., 2004; Dhamodharan and Wadsworth, 1995) and microtubule-interacting proteins such as stathmin, the kinesin MCAK, and EB1 (Cassimeris, 1999; Moore and Wordeman, 2004; Ringhoff and Cassimeris, 2009; Rusan et al., 2001) as well as numerous clinically approved or experimental anti-mitotic drugs including the taxanes, vinca alkaloids, and colchicine-like compounds modulate microtubule dynamic in cells (Jordan, 2002; Jordan and Kamath, 2007). In this chapter, we describe methods to analyze the dynamic instability of microtubules in living cells by microscopy of microinjected or expressed fluorescent tubulin, time-lapse microscopy, and analysis of time-dependent microtubule length changes.

Determination of drug binding to microtubules in vitro.

Many naturally occurring compounds and their synthetic analogs bind to soluble tubulin or to tubulin in microtubules. These compounds are often important drugs or drug candidates. They can potently alter both the dynamics and the polymer mass of microtubules. The binding affinity of a drug for soluble tubulin heterodimers is a common and relatively readily determined biochemical characteristic of a tubulin-targeted drug. However, it is only one important aspect of the drug-tubulin interaction. In solution and in cells, soluble tubulin is in equilibrium with polymerized microtubules. It is as important to determine drug binding to microtubules as it is to determine binding to soluble tubulin, since drug binding to microtubules frequently alters their function. The affinity of a compound for microtubules often differs vastly from its affinity for soluble tubulin. Here, we present detailed instructions for assessing binding stoichiometry and affinity to assembled unstabilized microtubules using radiolabeled drug. In addition, using examples from binding results with several important drugs including vinca alkaloids, colchicine, eribulin, and taxanes, we discuss aspects of the interactions with microtubules that may alter the experimental design of the drug-binding experiments.