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Research Techniques Made Simple: Preclinical Development of Combination Antitumor Targeted Therapies in Dermatology

      The identification and application of targeted therapies that inhibit critical pathways in malignant cells have shown tremendous promise for improving clinical outcomes for patients with advanced cutaneous malignancies. However, tumor cell heterogeneity, development of drug resistance, and risks of off-target effects remain barriers to prolonged remission and definitive cure. Herein, we describe the potential that combinations of antitumor targeted agents may offer in overcoming these challenges and detail techniques whereby promising combination regimens can be identified and further evaluated preclinically. Cancer cell lines and primary patient-derived malignant cells can be utilized to perform dose-response screenings in vitro for individual targeted agents before moving toward the evaluation of potential synergistic combinations. Mathematical analyses, including the Chou-Talalay method, determine combination indices and Hill slopes that permit relative comparisons among various drug combinations by quantification of synergistic activities. Further preclinical in vivo evaluation of promising single versus combination regimens may be studied in relevant mouse models of cutaneous malignancy. Ultimately, the formulation of combination targeted therapy regimens may be more broadly effective and less toxic, helping to better inform clinical trial design and prioritization.

      Abbreviations:

      3D (three-dimensional), ATP (adenosine triphosphate), AUC (area under the curve), CI (combination index), CTCL (cutaneous T-cell lymphoma), GI50 (growth inhibition at 50%), IC50 (inhibitory concentration at 50%), MEK (MAPK/ extracellular signal–regulated kinase kinase), PS (phosphatidyl-serine), SCC (squamous cell carcinoma), SMI (small-molecule inhibitor)

      Introduction

      We are experiencing an upsurge of pharmaceutical development in dermatology, including for cutaneous malignancies. Along with growing recognition of the specific activated and altered pathways within tumor cells, innovation in automated high-throughput screening has accelerated the discovery of new targeted therapies. However, tumor cell heterogeneity—both within a single tumor and between tumors from different patients—has proven that broadly applicable therapies for the majority of patients remain elusive. Despite the potential for personalized medicine approaches to further pair targeting drugs with aberrant pathways for each patient’s cancer, development of drug resistance and off-target effects may be limiting. Indeed, there remains a need for therapeutic regimens that are more broadly effective and that increase the potential for complete tumor resolution, prolonged remission, and cure. Utilizing drug combinations with synergistic activities against tumor cells is a promising strategy to increase therapeutic efficacy while potentially lowering effective drug concentrations. Herein, we describe techniques for preclinical screening, discovery, and validation of combination antitumor targeted agents that may have the potential for novel clinical applications in the treatment of advanced (i.e., unresectable, aggressive, metastatic) forms of cutaneous malignancy.

      Summary Points

      What information does this assay or technique provide?

      • Combination antitumor targeted therapies that exhibit synergy may increase the potential for effectiveness across varied tumor subtypes and delay development of resistance.
      • Automated, high-throughput, dose-response evaluation allows the screening of hundreds to thousands of candidate targeted agents, alone or in combination, against cancer cell lines or patient-derived malignant cells.
      • Drug combinations may have additive, antagonistic, synergistic, or potentiation effects as determined by mathematical models including Loewe additivity, Bliss independence, or Chou-Talalay models.
      • Animal models of cutaneous malignancy may be utilized for in vivo toxicity and effectiveness preclinical evaluation of promising combination regimens.

      Limitations

      • There are fundamental differences between established cancer cell lines and patient-derived malignant cells, and neither perfectly recapitulate the behavior of malignant cells in vivo.
      • Differing biology between mice and humans limits the applicability of mouse models.

      Materials and equipment for high-throughput drug screening

      Recent innovation in high-throughput drug screening has accelerated the in vitro screening of potential small-molecule inhibitors (SMIs) to target critical pathways in cancer cells. This process requires a tumor cell source; panels of SMI agents typically plated in microwell plates; and a series of specialized equipment that provides for automated, acoustic (nontouch), microfluidic transfers between the tumor cell and drug source plates (Figure 1). Tumor cells may derive from either established tumor cell lines or from patient-derived tumor cell short-term cultures, each with their advantages and disadvantages for drug screening. Established tumor cell lines may be passaged in vitro, frozen and stored for later use, and transplanted into syngeneic and/or immune-compromised laboratory animals to grow as tumors. Limitations to the use of passaged tumor cell lines include the potential for further development and selection of mutations in vitro, and that limited lines may not reflect the heterogeneity of disease in patients (
      • Gillet J.P.
      • Varma S.
      • Gottesman M.M.
      The clinical relevance of cancer cell lines.
      ;
      • Yumeen S.
      • Mirza F.N.
      • Lewis J.M.
      • King A.L.O.
      • Kim S.R.
      • Carlson K.R.
      • et al.
      JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL.
      ). Alternatively, primary tumor cell isolates from patients may be short-term cultured, and their genomic and transcriptional profiles are likely a more accurate representation of the spectrum of disease. However, such primary tumor cell lines may have limited survival in culture. Established or primary tumor cell lines can be cultured in two-dimensional cultures (e.g., in flasks) or in three-dimensional (3D) cultures using matrix or scaffolding to emulate organ structure. Such 3D artificial skin models can include various cell types such as keratinocytes and fibroblasts, thus better recapitulating the in vivo tumor cell microenvironments and organization.
      Figure thumbnail gr1
      Figure 1High-throughput evaluation of candidate combinations of antitumor targeted agents. Panels of single and combination antitumor targeted agents can be screened in vitro for their ability to kill malignant cells. Primary patient-derived cells or cancer cell lines are dispensed into microwell plates. Hundreds to thousands of candidate agents, alone or in combination, are dispensed via automated transfer into tumor cell–containing wells and coincubated. Tumor cell viability assays are used to create dose-response curves. (Created with biorender.com.) ATP, adenosine triphosphate; hr, hour; IC50, inhibitory concentration at 50%.
      SMI agents are potential therapeutic agents that can target and inhibit specific tumor cell pathways, most often by binding to key functional proteins involved in tumor cell signaling or proliferation. Inhibitory agents are generally smaller than 500 kDa, allowing them to diffuse across cell membranes to interact with intracellular proteins. SMIs may be specifically designed to target mutated gene products, such as BRAF inhibitors used to target the BRAF V600E protein common in malignant melanoma. Such mutation-specific SMIs may not be as useful, however, when driver mutations are heterogenous across patients, such as in cutaneous T-cell lymphoma (CTCL). High-throughput screening drug panels can be employed to discover SMI compounds with antitumor activity. Among the most commonly used SMI panels are kinase and phosphatase libraries, pharmaceutical industry candidates, natural products (e.g., isolated from rare plants), and current Food and Drug Administration–approved agents available for repurposing. Common antitumor cell pathways targeted include mediators and regulators of cell signaling, proliferation, apoptosis, and gene expression (Figure 2).
      Figure thumbnail gr2
      Figure 2Commonly targeted signaling pathways. Abnormal proliferation signals can result in malignant transformation and inappropriate proliferation of cells. Kinases and phosphatases along this pathway can be targeted to halt inappropriate signaling. Survival of malignant cells can also result when there are too many antiapoptotic signals and too few proapoptotic signals; thus, antiapoptotic proteins can also be targeted by SMIs. Transcription factors regulate expression of genes that are involved in any of the pathways that result in malignant cell survival and proliferation. Chromatin organization can be altered in malignant cells to result in an aberrant transcriptional profile, and thus DNA methylation or histone deacetylation can also be targeted. (Created with biorender.com and adapted with permission from
      • Yumeen S.
      • Mirza F.N.
      • Lewis J.M.
      • King A.L.O.
      • Kim S.R.
      • Carlson K.R.
      • et al.
      JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL.
      ). GPCR, G-protein coupled receptor; HDAC, histone deacetylase; SMI, small molecule inhibitor; STAT, signal transducer and activator of transcription; TRAIL, TNF-related apoptosis-inducing ligand.

      High-throughput drug screening

      Once candidate compounds are selected based on preliminary data, review of literature, or high-throughput preliminary screening (
      • Bell M.
      • Webster L.
      • Woodland A.
      Research techniques made simple: an introduction to drug discovery for dermatology.
      ), drug–tumor cell dose-response screening is performed. Equal numbers of tumor cells (e.g., 4,000–20,000 cells/well) are plated and incubated with increasing concentrations of candidate agents. Positive controls (i.e., 10% DMSO) that will cause death of all cells in the well (100% kill) and negative controls (i.e., vehicle alone) in which there is minimal cell death (0% kill) are used as reference controls. Cells are incubated with the candidate agents long enough to result in cell death (e.g., 24, 48, and/or 72 hours) but not so long that a significant portion of the negative control cells die. This generates dose-response curves from which inhibitory concentrations can be calculated. The inhibitory concentration at 50% (IC50) is considered the concentration of drug that results in half maximal kill of the cancer cells, but other more stringent inhibitory concentrations (e.g., 95% inhibitory concentration) may be used. Growth inhibition at 50% (GI50) can be used to assess cytostatic agents. In cases for which the IC50, GI50, or other value are challenging to determine, the area under the curve (AUC) can also provide information on a drug’s potency. The lower the AUC for a survival curve, the more potent the inhibitor. Hill slope (or slope factor) reflects the steepness of a dose-response curve and can provide an indication of ligand-receptor interaction between the inhibitor and its target.
      Several assays may be used to quantify cell viability and investigate potential mechanisms of inducing tumor cell death. Adenosine triphosphate (ATP)-release assays (e.g., CellTiter-Glo, Promega, Madison, WI) measures the amount of ATP present as an indicator of metabolically active cells. Remaining live cells are lysed with a detergent, releasing ATP into the medium that can be fluorometrically measured to quantify the number of living cells remaining. Similarly, cell metabolism assays, such as tetrazolium reduction cell viability, utilize a positively charged tetrazolium dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) that crosses the cell membrane in living cells and is converted into an insoluble purple-colored formazan product that can be quantified to determine the number of living cells.
      Induced tumor cell death can be further mechanistically characterized. Apoptosis may be intrinsic (driven by caspase-9) and/or extrinsic (driven by caspase-8). Both intrinsic and extrinsic apoptosis require activity of caspases 3 and 7; thus, their presence and activity are used as indicators of total apoptosis. In caspase-release assays (e.g., Caspase-Glo, Promega), lysed tumor cells release activated caspases that cleave a proluminescent substrate specific to the caspase of interest. Apoptosis and necrosis may be distinguished using fluorophore-conjugated annexin V that binds to phosphatidyl-serine (PS) residues that are upregulated on the surface of cells undergoing apoptosis. Conversely, cells undergoing necrosis lose integrity of the cell membrane, so annexin V can bind to intracellular PS, marking them as necrotic cells. For cytostatic agents without the capacity to induce cell death, quantification of cessation of proliferation (e.g., via Ki-67 expression or cell counts over time,) is alternatively utilized.

      Analyzing agent combinations for synergistic activity

      Putative drug combinations are evaluated to identify combinations of agents that are more effective when used together than either single agent (Figure 3a). Drug combination effects on tumor cell killing may be characterized as additive, antagonistic, or synergistic. If the tumor cell cytotoxic effect of two agents is additive, the effect of the combination of two drugs may simply be considered as the sum of their individual effects (% Kill Combination (Drug-A and Drug-B) = % Kill Drug-A + % Kill Drug-B). Antagonistic activity for drug combinations results in a lower percent kill than would be expected by the sum of their individual activities (% Kill Combination (Drug-A and Drug-B) < % Kill Drug-A + % Kill Drug-B). Synergistic activity is observed when a combination of agents results in a higher percent kill than would be expected with simple additive activity (% Kill Combination (Drug-A and Drug-B) > % Kill Drug-A + % Kill Drug-B), suggesting that such agents may be interacting (i.e., on the same or complementary pathways) to result in a greater induction of cell death than would be expected by purely additive effects of each individual agent. Potentiation (or enhancement) is described when one agent (Drug-A) has little effect on tumor cell viability alone, yet when paired with another agent (Drug-B), the combination results in a percent kill greater than that caused by Drug-B alone (% Kill Combination (Drug-A and Drug-B) > % Kill Drug-A (0) + % Kill Drug-B). This is expressed as a fold-increase in percent kill by the combination relative to Drug-B alone.
      Figure thumbnail gr3
      Figure 3Combination drug interactions. (a) Schematic dose-response curves showing the additive, antagonistic, synergistic, or potentiation activity of combinations of agents. (b) Chou-Talalay method isobolograms may be used to depict CIs of combination antitumor targeted therapies. A line representing the additive activity determined by the Chou-Talalay equation delineates CI points below the line indicating synergistic interactions and above the line indicating antagonistic activity. By plotting CI values collected by assessing drug combinations at a constant ratio, a curve can be extrapolated showing the relationship between % kill (fraction affected) and CI. This curve can be used to extrapolate CI at 90% kill to compare multiple drug combinations. CI, combination index.

      Mathematical models for evaluating drug cooperative activity

      Several mathematical models are utilized to evaluate the effectiveness of agents when used in combination, including quantification of the desired additive and synergistic effects on tumor cell cytotoxicity. In the Loewe additivity model, the median effect equation is used to calculate the expected effect of the combination of drugs, and a combination index (CI) is calculated. CIs < 1 are synergistic, whereas CIs > 1 indicate antagonism (
      • Loewe S.
      The problem of synergism and antagonism of combined drugs.
      ). This model provides the basis for the now more commonly used Chou-Talalay method. In the Bliss independence model, the observed response to the drug combinations is compared with a predicted response that is determined by multiplying the responses of the single agents (Expected % Kill Combination (Drug-A and Drug-B) = % Kill Drug-A × % Kill Drug-B). Any response greater than the predicted response is synergistic, and any response less than this value is antagonistic. A limitation of this model is that it cannot be used for those cases in which the two drugs may interact with one another in some way (
      • Zhao W.
      • Sachsenmeier K.
      • Zhang L.
      • Sult E.
      • Hollingsworth R.E.
      • Yang H.
      A new Bliss independence model to analyze drug combination data.
      ).
      Using the IC50 value and Hill slope from single-drug dose-response curves, the Chou-Talalay method further calculates CIs that may be interpreted similarly to the Loewe additivity model. The lower the CI values, the more synergistic the drug combination. For the same combination of drugs, CIs can vary based on concentrations of drug used and the resulting percent kill. Ideally, drug pairs should be evaluated at several concentrations and ratios. CIs calculated using constant ratio concentrations can be used to extrapolate a curve of CIs and kill percentages and extrapolate a CI at 90% kill (Figure 3b) that is considered to be clinically relevant and that allows for comparisons among candidate drug pairs (
      • Chou T.C.
      Drug combination studies and their synergy quantification using the Chou-Talalay method.
      ). Online Chou-Talalay method calculators are available at combosyn.com.

      Evaluating synergy in mouse models

      Once targeted antitumor combinations have been selected and evaluated in vitro, the next step in their preclinical development is to perform in vivo experiments. Mouse models have been developed for a number of cutaneous malignancies, including basal cell carcinoma, squamous cell carcinoma (SCC), melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, giant cell fibroblastoma, angiosarcoma, and CTCL, among others (
      • Gober M.D.
      • Bashir H.M.
      • Seykora J.T.
      Reconstructing skin cancers using animal models.
      ;
      • Verhaegen M.E.
      • Mangelberger D.
      • Weick J.W.
      • Vozheiko T.D.
      • Harms P.W.
      • Nash K.T.
      • et al.
      Merkel cell carcinoma dependence on bcl-2 family members for survival.
      ). These mouse models are typically created by targeted mutagenesis, upregulating or mutating genes known to be critical to the pathogenesis of these malignancies. A major limitation of such models is the ability to accurately reflect the heterogeneity of human malignancy, for which malignant transformation can involve multiple differing genes between individual patients.
      Alternatively, tumors can be induced in healthy mice by transplanting or injecting malignant cells (e.g., subcutaneously to form solid tumors in the skin or intravenously to maintain a clonal population in the blood as in Sézary syndrome). Human tumor cell lines or primary patient-derived cells can be injected into mice. However, humanized and/or immunocompromised (severe combined immunodeficiency, nude) mice must be used if injecting human cells (
      • Griffin R.L.
      • Kupper T.S.
      • Divito S.J.
      Humanized mice in dermatology research.
      ), and the inability to evaluate immune-based therapies or immunogenic cell death induction by cytotoxic agents in such immunodeficient mice remains a major limitation. Response to administered therapies can be measured by tumor incidence at a given timepoint or tumor response to decrease tumor burden. Therapies can be administered either locally (e.g., intratumoral injection) or systemically (e.g., intraperitoneally, intravenously, or by oral gavage). Advantages of mouse models of therapy include the potential for evaluation of combination antitumor therapies in a model that allows malignant cells to interact with their microenvironment and the host immune system in vivo (albeit without the immune influence in immunocompromised mouse models). Furthermore, the development of targeted effects and toxicities can be evaluated for single agents and combinations of agents in the same models but would still require major considerations around dosing to extrapolate the results from preclinical investigations for eventual clinical use. Limitations include differing biology between mice and humans, as well as the fundamental differences between established cancer cell lines and patient-derived malignant cells, in that neither perfectly recapitulates the natural state.

      Conclusions and clinical application

      With a greater understanding of the cellular and molecular mechanisms underpinning dermatologic malignancies, major strides have been taken toward improved targeted therapies, for example, BRAF and MAPK/extracellular signal–regulated kinase kinase (MEK) inhibitors for melanoma, EGFR inhibitors for SCC, and histone deacetylase and retinoid X receptor inhibitors for CTCL. Identification of key cancer pathways and the capacity to rapidly develop targeted therapeutic agents have accelerated cancer therapy discovery. However, emergence of drug resistance and resultant tumor recurrence, the lack of general applicability across varied tumor types, and dose-related toxicity and development off-target effects remain limiting factors to single-agent treatment. Heterogeneity in driver mutations across patient tumors, while lending well to precision medicine approaches, indicated that treatments that are broadly applicable to the majority of patients remain elusive.
      Herein, we described the potential of combination antitumor targeted agents in overcoming these challenges and outlined the techniques whereby combination regimens can be developed and evaluated preclinically. Preclinical evaluation may foster the identification and prioritization of promising combination regimens with the major objectives of increasing efficacy while decreasing off-target effects and development of resistance. Such combinations nonetheless would still necessitate major evaluation and validation clinically. We have recently utilized these methods for preclinical development of combination therapies for CTCL in vitro (
      • Mirza F.N.
      • Yumeen S.
      • Lewis J.M.
      • King A.L.O.
      • Kim S.R.
      • Carlson K.R.
      • et al.
      Screening novel agent combinations to expedite CTCL therapeutic development [e-pub ahead of print].
      ;
      • Yumeen S.
      • Mirza F.N.
      • Lewis J.M.
      • King A.L.O.
      • Kim S.R.
      • Carlson K.R.
      • et al.
      JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL.
      ). We found that the combination of Jak inhibition and BCL2 inhibition shows synergistic activity in vitro and that combinations of natural and over-the-counter compounds, including gentian violet and salinomycin, may be effective in synergistic combinations (
      • Mirza F.N.
      • Yumeen S.
      • Lewis J.M.
      • King A.L.O.
      • Kim S.R.
      • Carlson K.R.
      • et al.
      Screening novel agent combinations to expedite CTCL therapeutic development [e-pub ahead of print].
      ;
      • Yumeen S.
      • Mirza F.N.
      • Lewis J.M.
      • King A.L.O.
      • Kim S.R.
      • Carlson K.R.
      • et al.
      JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL.
      ).
      Follow on preclinical and clinical evaluation of such combinations discovered in vitro can further the development of combination regimens in hopes of increasing cure rates for advanced dermatologic malignancies. For example, BRAF inhibitors are combined with MEK inhibitors as the standard of treatment for patients with stage IV malignant melanoma that are positive for the BRAF V600E mutation, as this combination of SMIs has been shown to have synergistic antitumor activity, reduced toxicity, and delayed development of resistance (
      • Eroglu Z.
      • Ribas A.
      Combination therapy with BRAF and MEK inhibitors for melanoma: latest evidence and place in therapy.
      ;
      • Long G.V.
      • Hauschild A.
      • Santinami M.
      • Atkinson V.
      • Mandalà M.
      • Chiarion-Sileni V.
      • et al.
      Adjuvant dabrafenib plus trametinib in stage III BRAF-mutated melanoma.
      ). Trials are underway to include kinase inhibitors as well for a possible a triple combination for treatment of malignant melanoma.
      Although our review herein has focused on screening techniques utilizing SMIs, advances in genetic and genomic techniques have resulted in development of novel screening assays. These include CRISPR/Cas-9–based genomic screening, which can be applied to determine genes that, when suppressed in combination, exhibit synthetic lethality (
      • Huang A.
      • Garraway L.A.
      • Ashworth A.
      • Weber B.
      Synthetic lethality as an engine for cancer drug target discovery.
      ). Synthetic lethality occurs when two genes do not result in cancer cell death when suppressed alone, but when inhibited in combination, complementary pathways are inhibited, resulting in malignant cell death (
      • Huang A.
      • Garraway L.A.
      • Ashworth A.
      • Weber B.
      Synthetic lethality as an engine for cancer drug target discovery.
      ). Such screens may provide the basis for discovery of novel targets for combination treatments.
      Despite the promising nature of these screening techniques and strategies, there remain barriers to the efficient implementation of novel combination therapies for eventual usage in clinical settings. After preclinical identification and evaluation of drug combinations, repurposing agents that are already approved for other indications or using over-the-counter or natural compounds may accelerate further development. Combinations that may appear effective in in vitro or in vivo mouse models may nonetheless not exhibit synergy in translating to clinical practice. Nonetheless, the techniques and strategies outlined herein provide a means by which to prioritize novel combinations with the potential to address current challenges in treatment of dermatologic malignancies.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by the R. S. Evans Foundation and the Cutaneous Lymphoma Foundation.

      Author Contributions

      Conceptualization: SY, MG; Supervision: MG; Visualization: SY; Writing - Original Draft Preparation: SY, MG; Writing - Review and Editing: SY, FNM, JML, MG

      Multiple Choice Questions

      • 1.
        The potential advantages of utilizing combination antitumor targeted agent therapy over single-agent therapy include which of the following?
        • a.
          Higher doses of drugs are typically required.
        • b.
          All drivers of proliferation are simultaneously targeted.
        • c.
          Tumor cells and normal cells are killed equally.
        • d.
          Development of drug resistance may be potentially averted or delayed.
      • 2.
        Synergistic activity of drug combinations occurs when:
        • a.
          The effect of the combination of two drugs is equal to the sum of their individual effects.
        • b.
          The combination of drugs results in a higher percent kill than would be expected by the sum of their individual effects.
        • c.
          The combination of drugs results in a lower percent kill than would be expected by the sum of their individual activities.
        • d.
          One drug has no effect on cell viability alone but, when combined with another drug, shows enhanced activity.
      • 3.
        Which of the following can be derived from drug dose-response curves to compare the potency of candidate combination regimens?
        • a.
          Rate of drug action with time
        • b.
          Inhibitory concentration at 50% and Hill slope
        • c.
          Activation concentration
        • d.
          Mechanism of drug action in combination
      • 4.
        Which of the following assays can be utilized for the high-throughput screening of thousands of agents, alone and in combination, for antitumor cell cytotoxicity?
        • a.
          Adenosine triphosphate–release assay
        • b.
          Chou-Talalay method
        • c.
          Color conversion assay
        • d.
          qPCR
      • 5.
        Fluorophore-bound annexin V binds to which of the following to evaluate for malignant cell death in vitro?
        • a.
          Phosphatidyl-serine residues on the surface of malignant cells undergoing apoptosis
        • b.
          Cellular DNA of malignant cells undergoing apoptosis
        • c.
          Caspase substrates in cells undergoing extrinsic apoptosis
        • d.
          FAS-ligands in cells that are undergoing necrosis

      Detailed Answers

      • 1.
        The potential advantages of utilizing combination antitumor targeted agent therapy over single-agent therapy include which of the following?
      • CORRECT ANSWER: d. Development of drug resistance may be potentially averted or delayed.
      • Novel approaches to combination antitumor targeted therapies provide a means whereby development of resistance may be potentially averted by simultaneously targeting multiple drivers of malignant cell behavior. For management of malignant melanoma, combinations of BRAF inhibitors and MAPK inhibitors have been shown to be more efficacious than BRAF inhibitors alone in a phase III clinical trial, and this combination has been suggested to delay development of resistance to BRAF inhibitors (
        • Lim S.Y.
        • Menzies A.M.
        • Rizos H.
        Mechanisms and strategies to overcome resistance to molecularly targeted therapy for melanoma.
        ).
      • 2.
        Synergistic activity of drug combinations occurs when:
      • CORRECT ANSWER: b. The combination of drugs results in a higher percent kill than would be expected by the sum of their individual effects.
      • Synergistic activity is defined as activity that is greater than would be expected with simple additive activity, and thus the percent kill is greater than would be expected with the sum of the individual effects of the drugs. Synergistic activity suggests that the drugs may be interacting (i.e., either by acting on the same or complementary pathways) to result in a greater induction of cell death. Answer choice d. is indicative of potentiation.
      • 3.
        Which of the following can be derived from drug dose-response curves to compare the potency of candidate combination regimens?
      • CORRECT ANSWER: b. Inhibitory concentration at 50% and Hill slope
      • The inhibitory concentration at 50% (IC50) is considered the concentration of drug which results in half maximal kill of the cancer cells. The lower the IC50, the more potent the antitumor effect. The larger the Hill slope value, the steeper the curve and the more potent the inhibitor. IC50 and Hill slope of dose-response curves of different drugs or combinations of drugs can be compared to evaluate relative potency against malignant cells in vitro.
      • 4.
        Which of the following assays can be utilized for the high-throughput screening of thousands of agents, alone and in combination, for antitumor cell cytotoxicity?
      • CORRECT ANSWER: a. Adenosine triphosphate–release assay
      • Adenosine triphosphate (ATP)-release assays quantify the amount of ATP present as an indicator of metabolically active (live) cells. Remaining live cells after washing are lysed with a detergent, releasing ATP into the medium, which can be fluorometrically quantified via a luciferase assay to quantify the number of living cells present.
      • 5.
        Fluorophore-bound annexin V binds to which of the following to evaluate for malignant cell death in vitro?
      • CORRECT ANSWER: a. Phosphatidyl-serine residues on the surface of malignant cells undergoing apoptosis
      • Cells that undergo apoptosis increase expression of phosphatidyl-serine (PS) on their surfaces. Annexin V is a protein that binds specifically to PS. Fluorophore-conjugated annexin V can be added to cell media and will bind to PS on the surface of cells undergoing apoptosis. Cells that undergo necrosis lose integrity of the cell membrane, so annexin V can enter these cells and bind to intracellular PS, marking cells undergoing necrosis.

      Supplementary Material

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