Utilization of Photoaffinity Labeling to Investigate Binding of Microtubule Stabilizing Agents to P-Glycoprotein and ß-Tubulin.

Photoaffinity labeling approaches have historically been used in pharmacology to identify molecular targets. This methodology has played a pivotal role in identifying drug-binding domains and searching for novel compounds that may interact at these domains. In this review we focus on studies of microtubule stabilizing agents of natural product origin, specifically taxol (paclitaxel). Taxol and other microtubule interacting agents bind to both P-glycoprotein (ABCB1), a drug efflux pump that reduces intracellular drug accumulation, and the tubulin/microtubule system. Both binding relationships modulate drug efficacy and are of immense interest to basic and translational scientists, primarily because of their association with drug resistance for this class of molecules. We present this body of work and acknowledge its value as fundamental to understanding the mechanisms of taxol and elucidation of the taxol pharmacophore. Furthermore, we highlight the ability to multiplex photoaffinity approaches with other technologies to further enhance our understanding of pharmacologic interactions at an atomic level. Thus, photoaffinity approaches offer a relatively inexpensive and robust technique that will continue to play an important role in drug discovery for the foreseeable future.

Structural Refinement of the Tubulin Ligand (+)-Discodermolide to Attenuate Chemotherapy-Mediated Senescence.

The natural product (+)-discodermolide (DDM) is a microtubule stabilizing agent and potent inducer of senescence. We refined the structure of DDM and evaluated the activity of novel congeners in triple negative breast and ovarian cancers, malignancies that typically succumb to taxane resistance. Previous structure-activity analyses identified the lactone and diene as moieties conferring anticancer activity, thus identifying priorities for the structural refinement studies described herein. Congeners possessing the monodiene with a simplified lactone had superior anticancer efficacy relative to taxol, particularly in resistant models. Specifically, one of these congeners, B2, demonstrated 1) improved pharmacologic properties, specifically increased maximum response achievable and area under the curve, and decreased EC50; 2) a uniform dose-response profile across genetically heterogeneous cancer cell lines relative to taxol or DDM; 3) reduced propensity for senescence induction relative to DDM; 4) superior long-term activity in cancer cells versus taxol or DDM; and 5) attenuation of metastatic characteristics in treated cancer cells. To contrast the binding of B2 versus DDM in tubulin, X-ray crystallography studies revealed a shift in the position of the lactone ring associated with removal of the C2-methyl and C3-hydroxyl. Thus, B2 may be more adaptable to changes in the taxane site relative to DDM that could account for its favorable properties. In conclusion, we have identified a DDM congener with broad range anticancer efficacy that also has decreased risk of inducing chemotherapy-mediated senescence. SIGNIFICANCE STATEMENT: Here, we describe the anticancer activity of novel congeners of the tubulin-polymerizing molecule (+)-discodermolide. A lead molecule is identified that exhibits an improved dose-response profile in taxane-sensitive and taxane-resistant cancer cell models, diminished risk of chemotherapy-mediated senescence, and suppression of tumor cell invasion endpoints. X-ray crystallography studies identify subtle changes in the pose of binding to ß-tubulin that could account for the improved anticancer activity. These findings support continued preclinical development of discodermolide, particularly in the chemorefractory setting.

Improved Dose-Response Relationship of (+)-Discodermolide-Taxol Hybrid Congeners.

(+)-Discodermolide is a microtubule-stabilizing agent with potential for the treatment of taxol-refractory malignancies. (+)-Discodermolide congeners containing the C-3'-phenyl side chain of taxol (paclitaxel) were synthesized based on computational docking models predicting this moiety would fill an aromatic pocket of ß-tubulin insufficiently occupied by (+)-discodermolide, thereby conferring improved ligand-target interaction. It was recently demonstrated, however, that the C-3'-phenyl side chain occupied a different space, instead extending toward the M-loop of ß-tubulin, where it induced a helical conformation, hypothesized to improve lateral contacts between adjacent microtubule protofilaments. This insight led us to evaluate the biological activity of hybrid congeners using a panel of genetically diverse cancer cell lines. Hybrid molecules retained the same tubulin-polymerizing profile as (+)-discodermolide. Since (+)-discodermolide is a potent inducer of accelerated senescence, a fate that contributes to drug resistance, congeners were also screened for senescence induction. Flow cytometric and transcriptional analysis revealed that the hybrids largely retained the senescence-inducing properties of (+)-discodermolide. In taxol-sensitive cell models, the congeners had improved dose-response parameters relative to (+)-discodermolide and, in some cases, were superior to taxol. However, in cells susceptible to senescence, EMax increased without concomitant improvements in EC50 such that overall dose-response profiles resembled that of (+)-discodermolide.

Structural Basis of Microtubule Stabilization by Discodermolide.

Microtubule-stabilizing agents (MSAs) are widely used in chemotherapy. Using X-ray crystallography we elucidated the detailed binding modes of two potent MSAs, (+)-discodermolide (DDM) and the DDM-paclitaxel hybrid KS-1-199-32, in the taxane pocket of ß-tubulin. The two compounds bind in a very similar hairpin conformation, as previously observed in solution. However, they stabilize the M-loop of ß-tubulin differently: KS-1-199-32 induces an M-loop helical conformation that is not observed for DDM. In the context of the microtubule structure, both MSAs connect the ß-tubulin helices H6 and H7 and loop S9-S10 with the M-loop. This is similar to the structural effects elicited by epothilone A, but distinct from paclitaxel. Together, our data reveal differential binding mechanisms of DDM and KS-1-199-32 on tubulin.

Resistance to discodermolide, a microtubule-stabilizing agent and senescence inducer, is 4E-BP1-dependent.

Discodermolide is a microtubule-stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Lentiviral-mediated re-expression of wild-type 4E-BP1 in AD32 cells increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Consistent with this, cell growth and response to discodermolide was confirmed in vivo using tumor xenograft models. Furthermore, reintroduction of a nonphosphorylatable mutant (Thr-37/46 Ala) of 4E-BP1 was able to partially restore sensitivity and enhance proliferation in AD32 cells, suggesting that these effects are independent of phosphorylation by mTORC1. Microarray profiling of AD32-resistant cells versus sensitive A549 cells, and subsequent unbiased gene ontology analysis, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most statistically significant classes of differentially expressed genes included p53 signaling, G2/M checkpoint regulation, and genes involved in the role of BRCA1 in the DNA damage response. Consistent with this, p53 protein expression was up-regulated and had increased nuclear localization in AD32 cells relative to parental A549 cells. Furthermore, the stability of p53 was enhanced in AD32 cells. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence.

Nanoparticle-Encapsulated Doxorubicin Demonstrates Superior Tumor Cell Kill in Triple Negative Breast Cancer Subtypes Intrinsically Resistant to Doxorubicin.

The effect of size and release kinetics of doxorubicin-nanoparticles on anti-tumor efficacy was evaluated in a panel of human cancer cell lines, including triple-negative breast cancer (TNBC) cells that frequently demonstrate resistance to doxorubicin. Different nano-formulations of sol-gel-based Doxorubicin containing nanoparticles were synthesized. Increased cell kill in chemoreffactory triple-negative breast cancer cells was associated with the smallest size of nanoparticles and the slowest release of Dox. Modeling of dose-response parameters in Dox-sensitive versus Dox-resistant lines demonstrated increased EMax and area under the curve in Dox-resistant mesenchymal TNBC cells, implying potentially favorable activity in this molecular subtype of breast cancer. Mesenchymal TNBC cells demonstrated a high rate of fluorescent bead uptake suggestive of increased endocytosis, which may partially account for the enhanced efficacy of Dox-np in this subtype. Thus, manipulation of size and release kinetics of this nanoparticle platform is associated with enhanced dose-response metrics and tumor cell kill in therapeutically recalcitrant TNBC cell models. This platform is easily customizable and warrants further exploration.

Potentiation of taxol efficacy and by discodermolide in ovarian carcinoma xenograft-bearing mice.

PURPOSE: To evaluate the drug combination of discodermolide and Taxol in human ovarian cancer cells and in an in vivo model of ovarian carcinoma. EXPERIMENTAL DESIGN: The combination index method was used to evaluate the interaction of Taxol and discodermolide in human ovarian SKOV-3 carcinoma cells. Data were correlated with alterations in cell cycle distribution and caspase activation. In addition, SKOV-3 xenograft-bearing mice were treated with either Taxol, discodermolide, or a combination of both drugs given concurrently to evaluate the antitumor efficacy and toxicity of this combination. The Matrigel plug assay and CD31 immunohistochemistry were done to assess antiangiogenic effects. RESULTS: Taxol and discodermolide interact synergistically over a range of concentrations and molar ratios that cause drug-induced aneuploidy in ovarian carcinoma cells. In SKOV-3 xenograft-bearing mice, the combination is significantly superior to either single agent, and induces tumor regressions without notable toxicities. Immunohistochemical analysis of CD31 and Matrigel plug analysis show decreased vessel formation in mice treated with the combination relative to either drug alone. CONCLUSIONS: The synergistic activity of Taxol and discodermolide in cells is most potent at drug concentrations that result in drug-induced aneuploidy rather than mitotic arrest. Moreover, in an animal model of ovarian carcinoma, this is a well-tolerated combination that induces tumor regressions and suppresses angiogenesis. These data confirm the potency of this combination and support the use of concurrent low doses of Taxol and discodermolide for potential use in cancer therapeutics.

Enhancement of the therapeutic efficacy of taxol by the mitogen-activated protein kinase kinase inhibitor CI-1040 in nude mice bearing human heterotransplants.

Taxol may contribute to intrinsic chemoresistance by activating the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cytoprotective pathway in human cancer cell lines and tumors. We have previously shown additivity between Taxol and the MEK inhibitor, U0126 in human cancer cell lines. Here, the combination of Taxol with an orally bioavailable MEK inhibitor, CI-1040, was evaluated in human lung tumors heterotransplanted into nude mice. Unlike xenograft models that are derived from cells with multiple genetic alterations due to prolonged passage, heterotransplanted tumor models are more clinically relevant. Combined treatment with both drugs resulted in inhibition of tumor growth in all models and tumor regressions in three of four models tested, supporting our previous observation that Taxol's efficacy is potentiated by MEK inhibition. Concurrent administration was superior to intermittent dosing. Pharmacodynamic assessments of tumors indicated that suppression of MEK was associated with induction of S473 phosphorylated Akt and reduced proliferation in the combination groups relative to single agents, in addition to suppression of fibroblast growth factor-mediated angiogenesis and reduced expression of vascular endothelial growth factor. These findings are significant and indicate that this combination may have broad therapeutic applications in a diverse range of lung tumors with different intrinsic chemosensitivities.

Therapeutic Advances and New Directions for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a molecularly diverse grouping with poor prognosis for which chemotherapy remains the foundation of treatment. The molecular heterogeneity of the disease rationalizes its diverse biological behavior and differential response to treatment. Estimates of up to 20% of patients diagnosed have germline mutations in DNA-damage repair-pathway genes, namely BRCA1 and 2, and this can be used to select patients likely to respond to platinums and/or inhibitors of poly(ADP-ribose) polymerase (PARP). Similar strategies can be utilized in other subtypes of TNBC that have 'BRCA-like' tumor biology due to the presence of mutations in alternate DNA-damage repair genes. The diverse biological behavior of TNBC and its variable response to chemotherapy were largely decoded following genotyping studies that enabled the identification of distinct molecular subtypes, such that the biological and genetic heterogeneity of the disease could be understood. This subsequently enabled the identification of therapeutic 'vulnerabilities' for each subtype that encompass biological processes including proliferation, DNA repair, apoptosis, angiogenesis, immune modulation, and invasion and metastasis. To expedite the development of therapies for high-risk, early-stage breast cancer, we have adopted novel trial designs and re-defined endpoints as surrogates of clinical outcomes. The purpose of this review is to highlight the current standard and experimental treatment options for TNBC.

A Novel Indication for Panobinostat as a Senolytic Drug in NSCLC and HNSCC.

Panobinostat (pano) is an FDA-approved histone deacetylase inhibitor. There is interest in evaluating alternate dosing schedules and novel combinations of pano for the treatment of upper aerodigestive and lung malignancies; thus we evaluated it in combination with Taxol, a chemotherapeutic with activity in both diseases. Dose-dependent synergy was observed in Non-Small Cell Lung Cancer (NSCLC) and Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines and was due to senescence rather than potentiation of cell death. Senescence occurred following cisplatin- or Taxol-treatment in cell lines from both cancer types and was associated with decreased histone 3 (H3) acetylation and increased Bcl-xL expression: the latter a biomarker of senescence and target of anti-senescence therapeutics, or senolytics. Since H3 acetylation and Bcl-xL expression were altered in senescence, we subsequently evaluated pano as a senolytic in chemotherapy-treated cancer cells enriched for senescent cells. Pano caused cell death at significantly higher rates compared to repeat dosing with chemotherapy. This was associated with decreased expression of Bcl-xL and increased acetylated H3, reversing the expression patterns observed in senescence. These data support evaluating pano as a post-chemotherapy senolytic with the potential to kill persistent senescent cells that accumulate during standard chemotherapy in NSCLC and HNSCC.

Validation of the pharmacodynamics of BMS-247550, an analogue of epothilone B, during a phase I clinical study.

The primary aims of this study were to evaluate the timecourse and dose response of microtubule bundle formationin peripheral blood mononuclear cells (PBMCs) and to correlate these data with BMS-247550 pharmacokinetics. The data presented here were obtained from 17 patients enrolled in a Phase I trial who received five dose levels of BMS-247550 (7.4-59.2 mg/m(2)), given as a 1-h infusion once every 3 weeks. Plasma drug exposure or area under the curve (AUC), and tubulin bundle formation in PBMCs were assessed in cycles 1 and 2. Similar analyses were also performed on tumor biopsies from one eligible patient. PBMCs exhibited dramatic microtubule bundle formation 1 h after infusion that declined by 24 h, showing a positive correlation with AUC((0-24)) for cycles 1 and 2. A similar pattern of tubulin bundle formation also was observed in a smaller proportion of breast tumor cells from one patient who exhibited a partial response to BMS-247550. This patient's tumor expressed multidrug resistance (MDR1) and MDR-associated protein (MRP1), and in addition poly(ADPribose) polymerase cleavage, a marker of cell death, was observed within 23 h after drug infusion. This patient was also heterozygous for a novel polymorphism at the extreme COOH terminus of beta-tubulin (Gly 437 Gly/Ser), although the relevance of the polymorphism to the response is unknown. In summary, microtubule bundle formation in PBMCs occurs within 1 h of treatment with BMS-247550 and is related to plasma AUC. Similar bundle formation was seen in one tumor sample, despite expression of MDR1 and MRP1. Cell death occurred 23 h after peak microtubule bundle formation in these tumor cells. These findings validate in vitro pharmacodynamic observations.

Therapies for triple negative breast cancer.

INTRODUCTION: Triple negative breast cancer (TNBC) is a heterogeneous disease associated with a high risk of recurrence, and therapeutic options are currently limited to cytotoxic therapy. Germ-line mutations may occur in up to 20% of unselected patients with TNBC, which may serve as a biomarker identifying which patients may have tumors that are particularly sensitive to platinums and/or inhibitors of poly(ADP-ribose)polymerase. A substantial proportion of patients with TNBCs not associated with germ-line BRCA mutations may have tumors that are 'BRCA-like', rendering those individuals potential candidates for similar strategies. AREAS COVERED: The purpose of this review is to highlight the current standard and experimental treatment strategies. EXPERT OPINION: Recent research that has illuminated the molecular heterogeneity of the disease rationalizes its diverse biological behavior and differential response to chemotherapy. Modern technology platforms provide molecular signatures that can be mined for therapeatic interventions. Target pathways that are commonly dysregulated in cancer cells control cellular processes such as apoptosis, proliferation, angiogenesis, DNA repair, cell cycle progression, immune modulation and invasion, and metastasis. Novel trial design and re-defined endpoints as surrogates to clinical outcome have been introduced to expedite the development of breakthrough therapies to treat high-risk early-stage breast cancer.

Exploiting MEK inhibitor-mediated activation of ERα for therapeutic intervention in ER-positive ovarian carcinoma.

While the clinical benefit of MEK inhibitor (MEKi)-based therapy is well established in Raf mutant malignancies, its utility as a suppressor of hyperactive MAPK signaling in the absence of mutated Raf or Ras, is an area of ongoing research. MAPK activation is associated with loss of ERα expression and hormonal resistance in numerous malignancies. Herein, we demonstrate that MEKi induces a feedback response that results in ERα overexpression, phosphorylation and transcriptional activation of ER-regulated genes. Mechanistically, MEKi-mediated ERα overexpression is largely independent of erbB2 and AKT feedback activation, but is ERK-dependent. We subsequently exploit this phenomenon therapeutically by combining the ER-antagonist, fulvestrant with MEKi. This results in synergistic suppression of tumor growth, in vitro and potentiation of single agent activity in vivo in nude mice bearing xenografts. Thus, we demonstrate that exploiting adaptive feedback after MEKi can be used to sensitize ERα-positive tumors to hormonal therapy, and propose that this strategy may have broader clinical utility in ERα-positive ovarian carcinoma.

High expression of class III ß-tubulin predicts good response to neoadjuvant taxane and doxorubicin/cyclophosphamide-based chemotherapy in estrogen receptor-negative breast cancer.

BACKGROUND: Expression of class ΙΙΙ ß-tubulin (ßΙΙΙ-tubulin) correlates with tumor progression and resistance to taxane-based therapies for several human malignancies including breast cancer. However its predictive value in a neoadjuvant setting in breast cancer remains unexplored. The objective of this explorative study was to determine whether ßΙΙΙ-tubulin expression in breast cancer correlated with pathologic characteristics and whether its expression was predictive of response to neoadjuvant chemotherapy. PATIENTS AND METHODS: We determined ßΙΙΙ-tubulin expression in 85 breast cancers, including 41 localized breast cancers treated with primary surgery and 44 treated with neoadjuvant chemotherapy before surgery. ßΙΙΙ-tubulin expression was evaluated by immunohistochemical methods and was correlated with pathologic characteristics and response to neoadjuvant chemotherapy using residual cancer burden (RCB) score. RESULTS: High ßΙΙΙ-tubulin expression was significantly associated with poorly differentiated high-grade breast cancers (P = .003) but not with tumor size, estrogen receptor (ER) status, or human epidermal growth factor receptor 2 (HER2)/neu overexpression. In ER(-) tumors treated with neoadjuvant chemotherapy, high ßΙΙΙ-tubulin expression was associated with a significantly greater likelihood of achieving a good pathologic response to chemotherapy as reflected by lower RCB scores (P = .021). CONCLUSION: This study reveals differential ßΙΙΙ-tubulin expression in breast cancers of different histologic grades, hormone receptors, and HER2/neu status. It also suggests a potential role for ßΙΙΙ-tubulin as a predictive biomarker for response in neoadjuvant chemotherapy for ER(-) breast cancer, which has not been previously reported. These data provide a strong rationale for considering ßΙΙΙ-tubulin status and further validation of this marker in a large study.

Characterization of a human ßV-tubulin antibody and expression of this isotype in normal and malignant human tissue.

There are seven distinct ß-tubulin isotypes and eight α-tubulin isotypes in mammals that are hypothesized to have tissue- and cell-specific functions. There is an interest in the use of tubulin isotypes as prognostic markers of malignancy. ßV-tubulin, like ßIII-tubulin, has been implicated in malignant transformation and drug resistance, however little is known about its localization and function. Thus, we generated for the first time, a rabbit polyclonal antibody specific for human ßV-tubulin. The antibody did not cross-react with mouse ßV-tubulin or other human ß-tubulin isotypes and specifically labeled ßV-tubulin by immunoblotting, immunofluorescence and immunohistochemistry. Immunohistochemistry of various human normal tissues revealed that ßV-tubulin was expressed in endothelial cells, myocytes and cells with muscle differentiation, structures with transport and/or secretory function such as renal tubules, pancreatic ducts and bile ducts, and epithelium with secretory function such as prostate. ßV-tubulin was also specifically expressed in pancreatic islets and intratubular germ cell neoplasia, where it may have diagnostic utility. Initial studies in breast, lung and ovarian cancers indicated aberrant expression of ßV-tubulin, suggesting that this isoform may be associated with tumorigenesis. Thus, ßV-tubulin expression is a potentially promising prognostic marker of malignancy.

Insights into 4E-BP1 and p53 mediated regulation of accelerated cell senescence.

Senescence is a valid tumor suppressive mechanism in cancer. Accelerated cell senescence describes the growth arrested state of cells that have been treated with anti-tumor drugs, such as doxorubicin that induce a DNA damage response. Discodermolide, a microtubule-stabilizing agent, is a potent inducer of accelerated cell senescence. Resistance to discodermolide is mediated via resistance to accelerated cell senescence, and is associated with reduced expression of the mTORC1 substrate, 4E-BP1 and increased expression of p53 [1]. Although the association of p53 with senescence induction is well-characterized, senescence reversion in the presence of high expression of p53 has not been well-documented. Furthermore, studies addressing the role of mTOR signaling in regulating senescence have been limited and recent data implicate a novel, senescence-associated role for 4E-BP1 in crosstalk with the transcription factor p53. This research perspective will address these somewhat contradictory findings and summarize recent research regarding senescence and mTORC1 signaling.

Targeting protein translation in human non small cell lung cancer via combined MEK and mammalian target of rapamycin suppression.

Lung cancer is a genetically heterogeneous disease characterized by the acquisition of somatic mutations in numerous protein kinases, including components of the rat sarcoma viral oncogene homolog (RAS) and AKT signaling cascades. These pathways intersect at various points, rendering this network highly redundant and suggesting that combined mitogen-activated protein/extracellular signal-regulated kinase (MEK) and mammalian target of rapamycin (mTOR) inhibition may be a promising drug combination that can overcome its intrinsic plasticity. The MEK inhibitors, CI-1040 or PD0325901, in combination with the mTOR inhibitor, rapamycin, or its analogue AP23573, exhibited dose-dependent synergism in human lung cancer cell lines that was associated with suppression of proliferation rather than enhancement of cell death. Concurrent suppression of MEK and mTOR inhibited ribosomal biogenesis by 40% within 24 h and was associated with a decreased polysome/monosome ratio that is indicative of reduced protein translation efficiency. Furthermore, the combination of PD0325901 and rapamycin was significantly superior to either drug alone or PD0325901 at the maximum tolerated dose in nude mice bearing human lung tumor xenografts or heterotransplants. Except for a PTEN mutant, all tumor models had sustained tumor regressions and minimal toxicity. These data (a) provide evidence that both pathways converge on factors that regulate translation initiation and (b) support therapeutic strategies in lung cancer that simultaneously suppress the RAS and AKT signaling network.