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Supplementary Fig. S1 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Purpose:The purpose of this study was to evaluate the rational combination of TORC1/2 inhibitor TAK-228 and Aurora A kinase inhibitor alisertib in preclinical models of triple-negative breast cancer (TNBC) and to conduct a phase I dose escalation trial in patients with advanced solid tumors.Experimental Design:TNBC cell lines and patient-derived xenograft (PDX) models were treated with alisertib, TAK-228, or the combination and evaluated for changes in proliferation, cell cycle, mTOR pathway modulation, and terminal cellular fate, including apoptosis and senescence. A phase I clinical trial was conducted in patients with advanced solid tumors treated with escalating doses of alisertib and TAK-228 using a 3+3 design to determine the maximum tolerated dose (MTD).Results:The combination of TAK-228 and alisertib resulted in decreased proliferation and cell-cycle arrest in TNBC cell lines. Treatment of TNBC PDX models resulted in significant tumor growth inhibition and increased apoptosis with the combination. In the phase I dose escalation study, 18 patients with refractory solid tumors were enrolled. The MTD was alisertib 30 mg b.i.d. days 1 to 7 of a 21-day cycle and TAK-228 2 mg daily, continuous dosing. The most common treatment-related adverse events were neutropenia, fatigue, nausea, rash, mucositis, and alopecia.Conclusions:The addition of TAK-228 to alisertib potentiates the antitumor activity of alisertib in vivo, resulting in increased cell death and apoptosis. The combination is tolerable in patients with advanced solid tumors and should be evaluated further in expansion cohorts with additional pharmacodynamic assessment.

Supplementary Fig. S2 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Methods from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Fig. S7 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Table S3 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Pharmacodynamic effects of alisertib and TAK-228 in patient tissue samples

Supplementary Fig. S8 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Fig. S4 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Fig. S6 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Table S2 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Kinesin-5 inhibitors (K5I) are promising antimitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using time-lapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and noncancer cell types to undergo prolonged arrest in monopolar mitosis. Subsequent events, however, differed greatly between cell types. Normal diploid cells mostly slipped from mitosis and arrested in tetraploid G1, with little cell death. Several cancer cell lines died either during mitotic arrest or following slippage. Contrary to prevailing views, mitotic slippage was not required for death, and the duration of mitotic arrest correlated poorly with the probability of death in most cell lines. We also assayed drug reversibility and long-term responses after transient drug exposure in MCF7 breast cancer cells. Although many cells divided after drug washout during mitosis, this treatment resulted in lower survival compared with washout after spontaneous slippage likely due to chromosome segregation errors in the cells that divided. Our analysis shows that K5Is cause cancer-selective cell killing, provides important kinetic information for understanding clinical responses, and elucidates mechanisms of drug sensitivity versus resistance at the level of phenotype. [Mol Cancer Ther 2008;7(11):3480–9]

Supplementary Fig. S5 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Fig. from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Fig. S3 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Table S1 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Video 1 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 1 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 2 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 2 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 1b from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Video 3 from A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization

Cells form transient, circular dorsal ruffles or “waves” in response to stimulation of receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor. These dynamic structures progress inward on the dorsal surface and disappear, occurring concomitantly with a marked reorganization of F-actin. The cellular function of these structures is largely unknown. Here we show that EGF-induced waves selectively sequester and internalize ∼50% of ligand-bound EGFR from the cell surface. This process requires receptor phosphorylation, active phosphatidylinositol 3-kinase, and dynamin 2, although clathrin-coated pits or caveolae are not required. Epithelial and fibroblast cells stimulated with EGF sequestered EGFR rapidly into waves that subsequently generated numerous receptor-positive tubular-vesicular structures. Electron microscopy confirmed that waves formed along the dorsal membrane surface and extended numerous tubules into the cytoplasm. These findings characterize a structure that selectively sequesters large numbers of activated EGFR for their subsequent internalization, independent of traditional endocytic mechanisms such as clathrin pits or caveolae. (Cancer Res 2006; 66(7): 3603-10)

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level. Cancer Res; 71(13); 4608–16. ©2011 AACR.

Supplementary Figure 3 from A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization

Supplementary Figure 2 from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Table 1 from A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization

Supplementary Figure and Video Legends 1-3 from A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization

Supplementary Video 7 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Methods from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 2 from A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization

Supplementary Video 1 from A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization

Supplementary Figure 1 from A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization

Supplementary Figure 2 from A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization

Supplementary Video 6 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 7b from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Figure Legends 1-7 from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Video 5 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 4 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 3 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 6a from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Figure Legends 1-4, Movie Legends 1-8 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

To improve cancer chemotherapy, we need to understand the mechanisms that determine drug sensitivity in cancer and normal cells. Here, we investigate this question across a panel of 11 cell lines at a phenotypic and molecular level for three antimitotic drugs: paclitaxel, nocodazole, and an inhibitor of kinesin-5 (also known as KSP, Eg5, Kif11). Using automated microscopy with markers for mitosis and apoptosis (high content screening), we find that the mitotic arrest response shows relatively little variation between cell types, whereas the tendency to undergo apoptosis shows large variation. We found no correlation between levels of mitotic arrest and apoptosis. Apoptosis depended on entry into mitosis and occurred both from within mitosis and after exit. Response to the three drugs strongly correlated, although paclitaxel caused more apoptosis in some cell lines at similar levels of mitotic arrest. Molecular investigations showed that sensitivity to apoptosis correlated with loss of an antiapoptotic protein, XIAP, during the drug response, but not its preresponse levels, and to some extent also correlated with activation of the p38 and c-Jun NH2 kinase pathways. We conclude that variation in sensitivity to antimitotic drugs in drug-naive cell lines is governed more by differences in apoptotic signaling than by differences in mitotic spindle or spindle assembly checkpoint proteins and that antimitotics with different mechanisms trigger very similar, but not identical, responses. [Cancer Res 2008;68(9):3269–76]

Supplementary Video 8 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 3 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 3a from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Figure 5a from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Table 4 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

The proteasome holoenzyme regulates the cellular proteome via degrading most proteins. In its 19-subunit regulatory particle (RP), a heterohexameric ATPase enables protein degradation by injecting protein substrates into the core peptidase. RP assembly utilizes "checkpoints," where multiple dedicated chaperones bind to specific ATPase subunits and control the addition of other subunits. Here, we find that the RP assembly checkpoint relies on two common features of the chaperones. Individual chaperones can distinguish an RP, in which their cognate ATPase persists in the ATP-bound state. Chaperones then together modulate ATPase activity to facilitate RP subunit rearrangements for switching to an active, substrate-processing state in the resulting proteasome holoenzyme. Thus, chaperones may sense ATP binding and hydrolysis as a readout for the quality of the RP complex to generate a functional proteasome holoenzyme. Our findings provide a basis to potentially exploit the assembly checkpoints in situations with known deregulation of proteasomal ATPase chaperones.

Purpose: The purpose of this study was to evaluate the rational combination of TORC1/2 inhibitor TAK-228 and Aurora A kinase inhibitor alisertib in preclinical models of triple-negative breast cancer (TNBC) and to conduct a phase I dose escalation trial in patients with advanced solid tumors. Experimental Design: TNBC cell lines and patient-derived xenograft (PDX) models were treated with alisertib, TAK-228, or the combination and evaluated for changes in proliferation, cell cycle, mTOR pathway modulation, and terminal cellular fate, including apoptosis and senescence. A phase I clinical trial was conducted in patients with advanced solid tumors treated with escalating doses of alisertib and TAK-228 using a 3+3 design to determine the maximum tolerated dose (MTD). Results: The combination of TAK-228 and alisertib resulted in decreased proliferation and cell-cycle arrest in TNBC cell lines. Treatment of TNBC PDX models resulted in significant tumor growth inhibition and increased apoptosis with the combination. In the phase I dose escalation study, 18 patients with refractory solid tumors were enrolled. The MTD was alisertib 30 mg b.i.d. days 1 to 7 of a 21-day cycle and TAK-228 2 mg daily, continuous dosing. The most common treatment-related adverse events were neutropenia, fatigue, nausea, rash, mucositis, and alopecia. Conclusions: The addition of TAK-228 to alisertib potentiates the antitumor activity of alisertib in vivo, resulting in increased cell death and apoptosis. The combination is tolerable in patients with advanced solid tumors and should be evaluated further in expansion cohorts with additional pharmacodynamic assessment.