Siang-Boon Koh

An automated fitting procedure and software for dose-response curves with multiphasic features

In cancer pharmacology (and many other areas), most dose-response curves are satisfactorily described by a classical Hill equation (i.e. 4 parameters logistical). Nevertheless, there are instances where the marked presence of more than one point of inflection, or the presence of combined agonist and antagonist effects, prevents straight-forward modelling of the data via a standard Hill equation. Here we propose a modified model and automated fitting procedure to describe dose-response curves with multiphasic features. The resulting general model enables interpreting each phase of the dose-response as an independent dose-dependent process. We developed an algorithm which automatically generates and ranks dose-response models with varying degrees of multiphasic features. The algorithm was implemented in new freely available Dr Fit software (sourceforge.net/projects/drfit/). We show how our approach is successful in describing dose-response curves with multiphasic features. Additionally, we analysed a large cancer cell viability screen involving 11650 dose-response curves. Based on our algorithm, we found that 28% of cases were better described by a multiphasic model than by the Hill model. We thus provide a robust approach to fit dose-response curves with various degrees of complexity, which, together with the provided software implementation, should enable a wide audience to easily process their own data.

Curcumin Analogues with Potent and Selective Anti‐proliferative Activity on Acute Promyelocytic Leukemia: Involvement of Accumulated Misfolded Nuclear Receptor Co‐repressor (N‐CoR) Protein as a Basis for Selective Activity

Curcumin arrests the proliferation of acute promyelocytic leukemia (APL) cells by stabilizing the misfolded nuclear receptor co‐repressor (N‐CoR) protein, thereby sensitizing APL cells to apoptosis induced by the unfolded protein response. This phenomenon was attributed to inhibition of the proteasomal and protease‐induced breakdown of misfolded N‐CoR by curcumin. Curcumin is, however, a modest inhibitor and affected the viability of APL cells at micromolar concentrations. Modifying curcumin at its conjugated β‐diketone linker and terminal phenyl rings yielded potent congeners with sub‐micromolar growth inhibitory activities which selectively kill APL cells over non‐APL leukemic and nonmalignant cells. Analogues with pronounced APL‐selective anti‐proliferative activities, as observed in representative dibenzylidenecyclohexanones and dibenzylidenecyclopentanones, strongly promoted the accumulation of misfolded and nonfunctional N‐CoR at significantly lower concentrations than their growth inhibitory IC50 values. These compounds also inhibited the human 20S proteasome in an enzyme‐based assay, thus providing convincing support for the prevailing hypothesis that impeding the degradation of N‐CoR is a key mechanistic event contributing to APL cell death.

A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level

ABSTRACT The fluorescence ubiquitination-based cell cycle indicator (FUCCI) is a powerful tool for use in live cells but current FUCCI-based assays have limited throughput in terms of image processing and quantification. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells by using an all-in-one expression cassette, FastFUCCI. The approach alleviated the need for sequential transduction and characterisation, improving labelling efficiency. We coupled the system to an automated imaging workflow capable of handling large datasets. The integrated assay enabled analyses of single-cell readouts at high spatiotemporal resolution. With the assay, we captured in detail the cell cycle alterations induced by antimitotic agents. We found that treated cells accumulated at G2 or M phase but eventually advanced through mitosis into the next interphase, where the majority of cell death occurred, irrespective of the preceding mitotic phenotype. Some cells appeared viable after mitotic slippage, and a fraction of them subsequently re-entered S phase. Accordingly, we found evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrate the utility of the FastFUCCI assay for quantifying spatiotemporal dynamics and identify its potential in preclinical drug development. Summary: This article presents the preclinical utility of an automated in vitro imaging platform that enables quantitative single-cell spatiotemporal monitoring of the cell cycle.

Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy

Highlights • A workflow for mitosis analysis in phase contrast microscopy is proposed.• It is derived from mathematical imaging concepts.• Those include the circular Hough transform and variational segmentation methods.• Results for live-cell imaging experiments are presented.

Modelling of the cancer cell cycle as a tool for rational drug development: A systems pharmacology approach to cyclotherapy

The dynamic of cancer is intimately linked to a dysregulation of the cell cycle and signalling pathways. It has been argued that selectivity of treatments could exploit loss of checkpoint function in cancer cells, a concept termed “cyclotherapy”. Quantitative approaches that describe these dysregulations can provide guidance in the design of novel or existing cancer therapies. We describe and illustrate this strategy via a mathematical model of the cell cycle that includes descriptions of the G1-S checkpoint and the spindle assembly checkpoint (SAC), the EGF signalling pathway and apoptosis. We incorporated sites of action of four drugs (palbociclib, gemcitabine, paclitaxel and actinomycin D) to illustrate potential applications of this approach. We show how drug effects on multiple cell populations can be simulated, facilitating simultaneous prediction of effects on normal and transformed cells. The consequences of aberrant signalling pathways or of altered expression of pro- or anti-apoptotic proteins can thus be compared. We suggest that this approach, particularly if used in conjunction with pharmacokinetic modelling, could be used to predict effects of specific oncogene expression patterns on drug response. The strategy could be used to search for synthetic lethality and optimise combination protocol designs.

Exploration and Optimization of Structure–Activity Relationships in Drug Design using the Taguchi Method

In the exploration of structure–activity relationships (SARs), current approaches are still heavily influenced by intuition and serendipity. A common approach to lead optimization in drug discovery is to vary one substitution site at a time, while keeping other sites in the molecule unchanged. This approach is described as the one-factor-at-a-time (OFAT) technique and is based on the underlying assumption that the effect of each substituent is isolated from that of other substituents—that is, its effect on the biological response is independent of responses elicited by substituents at other sites of the molecule. Accordingly, the design of analogues involving multiple substitution sites cannot be handled efficiently by the OFAT approach. For example, if the optimum group at one substitution site has been determined, one cannot assume that it will still be the preferred group when changes are made to other sites. Under such circumstances, it is said that there are interactions between the substitution sites. The problem with the OFAT approach is that it does not readily identify such interactions. Furthermore, OFAT is likely to overlook certain combinations of groups at the substitution sites. Thus, some compounds termed blind spot compounds will not be synthesized or evaluated for biological activity (Figure S1 in the Supporting Information). The question can then be asked if the choice of groups at each site in those compounds that are synthesized is indeed optimal, and hence if the resulting SARs have been correctly deduced. The decision-making approach adopted in OFAT tends to be subjective and could lead to the optimal compound being overlooked. The true SAR landscape is a combinatorial expansion of all the R groups at the various attachment points on the scaffold, but the resources and time required for the systematic exploration of the entire landscape are often overwhelming. Here, we propose a novel application of the Taguchi method as an alternative approach to the optimization of chemical modifications to a core structure. The Taguchi method, an approach based on the design of experiments (DoE) method, is widely used in the manufacturing industry for quality engineering purposes. In DoE, a design matrix is constructed in a combinatorial fashion that specifies the levels for several different factors for each experiment. A key feature of the Taguchi method is the orthogonal array, which is a predefined table specifying factors and levels. The simplest array (L4) is used to design experiments involving three factors with two levels per factor. In the lead modification context, an L4 array would investigate three substitution sites with two different substituents per site. In order to investigate interactions between sites and groups, four “experiments” (equivalent to synthesized compounds) are necessary. To evaluate the optimal variable settings, the Taguchi method recommends the use of the loss function as a means of monitoring the deviation in the quality characteristic. In the present context, the optimal variable settings are the preferred functionalities at the substitution sites, and the quality characteristic is the biological response elicited by the test compound, as reflected by its IC50 value, the concentration required to reduce a response (specific binding, enzyme activity, cell viability, etc.) to 50 % of that observed in the absence of the test compound (Table S1 in the Supporting Information). Here, the desired outcome of lead optimization is to reduce the magnitude of the quality characteristic (“the-lower-the-better”) since compounds with smaller IC50 values are more potent and are perhaps more likely to progress down the discovery pipeline. The deviation in the IC50 values is monitored as the signalto-noise (S/N) ratio. As this ratio measures the noise arising from repeated determinations of the IC50 value, a more positive/less negative value is desired. The S/N ratio (hs) for each compound (s) is determined using Equation (1), where yij is the IC50 or EC50 value of the ith replicate of the jth compound, and the summation i extends over the number of replicate measurements (n). Table 1 illustrates this process for the L4 Taguchi array.

The ATR inhibitor AZD6738 synergizes with gemcitabine in vitro and in vivo to induce pancreatic ductal adenocarcinoma regression

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, and overall survival rates have barely improved over the past five decades. The antimetabolite gemcitabine remains part of the standard of care but shows very limited antitumor efficacy. Ataxia telangiectasia and Rad3-related protein (ATR), the apical kinase of the intra–S-phase DNA damage response, plays a central role in safeguarding cells from replication stress and can therefore limit the efficacy of antimetabolite drug therapies. We investigated the ability of the ATR inhibitor, AZD6738, to prevent the gemcitabine-induced intra–S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo. In PDAC cell lines, AZD6738 inhibited gemcitabine-induced Chk1 activation, prevented cell-cycle arrest, and restrained RRM2 accumulation, leading to the strong induction of replication stress markers only with the combination. Moreover, synergistic growth inhibition was identified in a panel of 5 mouse and 7 human PDAC cell lines using both Bliss Independence and Loewe models. In clonogenic assays, the combination abrogated survival at concentrations for which single agents had minor effects. In vivo, AZD6738 in combination with gemcitabine was well tolerated and induced tumor regression in a subcutaneous allograft model of a KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse cancer cell line, significantly extending survival. Remarkably, the combination also induced regression of a subgroup of KPC autochthonous tumors, which generally do not respond well to conventional chemotherapy. Altogether, our data suggest that AZD6738 in combination with gemcitabine merits evaluation in a clinical trial in patients with PDAC. Mol Cancer Ther; 17(8); 1670–82. ©2018 AACR.

Abstract B19: The ATR inhibitor, AZD6738, synergizes with other DNA damage response inhibitors and genotoxic drugs in pancreatic ductal adenocarcinoma cell lines: Opportunities for new therapeutic combinations

Mutations in oncogenes, tumor suppressor and DNA damage response (DDR) mediator genes drive or permit malignant transformation but also increase endogenous replication stress. The serine/threonine kinase ATR plays a critical role in safeguarding genome integrity from such replication stress and several studies have demonstrated the increased reliance of cancer cells on ATR function. We investigated the therapeutic opportunities for the ATR inhibitor, AZD6738, in combination with DNA damaging or DDR-targeted agents, in the context of pancreatic ductal adenocarcinoma (PDAC). We evaluated four DNA-damaging agents (gemcitabine, 5-fluorouracil, oxaliplatin, SN38 (the active metabolite of irinotecan)) and three DDR-targeted agents (Wee1 inhibitor (AZD1775), Chk1 inhibitor (MK8776), PARP inhibitor (AZD2281)), each in combination with AZD6738 at multiple concentrations. Efficacy of these combinations was tested in growth inhibition assays in vitro, using a panel of cell lines in order to capture some of the genetic heterogeneity observed in PDAC: two human cell lines and four lines from the KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse. Synergistic growth inhibition was identified applying both Bliss Independence and Loewe models, using Combenefit software. All the KPC mouse cell lines were sensitive to AZD6738 as a single agent, with GI50 ranging from 346 to 566 nM. MIA PaCa-2 were sensitive to AZD6738, achieving >90% growth inhibition, with GI50 of 2.2 μM. PANC-1 cells were less sensitive, with GI50 21 μM and achieving only ~60% GI, at the highest concentration tested. PANC-1 cells are also less sensitive to gemcitabine than the other cell lines. Synergy was detected in most of the cell lines, with each of the seven drug combinations tested. The combinations of AZD6738 with gemcitabine and with oxaliplatin showed synergy in all cell lines tested. We next investigated scheduling of the gemcitabine/ATRi combination, at the specific GI50 concentrations for each cell line, using kinetic live-cell imaging assays. Concurrent treatment of gemcitabine/ATRi for 16h proved to be most effective, almost completely inhibiting cell growth for more than three days after washout. Sequential treatment (irrespective of the order) or shorter pulses (8h) were less effective. Maintaining ATRi after gemcitabine washout further enhanced growth inhibition for most cell lines. Mechanistically, ATRi impaired Chk1 activation (p-Ser345) and, in combination with gemcitabine, strongly potentiated DNA damage (gamma H2AX). Maintaining ATRi after gemcitabine washout helped to sustain the level of DNA damage. In vivo studies are underway to determine whether the gemcitabine/ATRi combination enhances efficacy compared to gemcitabine alone. The ATRi/oxaliplatin combination is also being investigated in vitro and in vivo using similar methods. Several genes have been described in the literature to increase the reliance on ATR functions when altered. Mining two published datasets (TCGA, 186 samples and UTSW, Nat. Commun . 2015, 109 samples) we have investigated the frequencies at which 21 of these genes were altered in human PDAC. Overall ~95% of PDAC samples exhibit at least one (9% only one, 28% two and 57% three or more) genetic alteration likely to sensitize to ATRi, potentially improving the therapeutic index of combination approaches. Thus, combinations including ATRi merit further evaluation as they have the potential to be effective in the treatment of patients with PDAC. Citation Format: Yann Wallez, Siang-Boon Koh, Venkata Sai Vivek Bhogadi, Alan Lau, Frances M. Richards, Duncan I. Jodrell. The ATR inhibitor, AZD6738, synergizes with other DNA damage response inhibitors and genotoxic drugs in pancreatic ductal adenocarcinoma cell lines: Opportunities for new therapeutic combinations [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B19.

Abstract B62: Evaluation of scheduling for triple therapy gemcitabine/CHEK1 inhibitor/WEE1 inhibitor in pancreatic cancer models

Sensitization of cancer cells to gemcitabine (Gem) has been shown with checkpoint kinase CHEK1 (CHK1) and WEE1 inhibitors. Our aim is to determine, using in vitro and in vivo models, the optimal regime for application of the triple combination in pancreatic cancer. First, we have investigated the dual combinations of Gem + either CHEK1 or WEE1 inhibitors in growth inhibition assays in vitro. Synergistic growth inhibition was identified using both Bliss Independence and Loewe models, in MIA PaCa-2 cells at 10 – 30 nM Gem + 300 – 1000 nM CHEK1 inhibitor MK8776; or 10 – 30 nM Gem + 30 – 100 nM WEE1 inhibitor MK1775. The synergistic concentrations were submaximal, i.e. below the single agent GI50 concentrations, which were 30 nM for Gem, 6 μM for MK8776, and 500 nM for MK1775. The Panc-1 cell line is more resistant to Gem as a single agent, but synergy was evident: in colony forming assays the dual combination of either 30 nM gem + 300 nM MK1775 or 30 nM Gem + 1 μM MK8776 inhibited colony formation by 97 +/- 1.6 % and 91 +/- 2% respectively. Single agent Gem inhibited colony formation by only 23 +/- 4 % and MK1775 or MK8776 did not inhibit colony formation at these concentrations. Next, we investigated scheduling of the Gem/CHEK1i/Wee1i triple combination, at the synergistic concentrations, with kinetic live-cell imaging assays. MIA PaCa-2 cells treated continuously with 10 nM Gem plus 1 μM MK8776 showed durable growth inhibition over 72 hours. However, if both drugs were washed off after 24 hours the Gem + CHEK1i-treated cells recovered and began to proliferate within 24 hours. Different schedules of the trio were tested, and the most durable growth inhibition (> 5 days) was obtained when Gem + MK8776 were washed off at 24 hours and replaced with 300 nM MK1775, whereas 300 nM MK1775 in the absence of Gem + MK8776 pretreatment did not significantly inhibit growth, compared to control. Different scheduling options were also tested in Panc-1 cells in colony forming assays, and again the most effective schedule was Gem + MK8776 for 24 hours, followed by MK1775. MIA PaCa-2 xenograft studies are now underway, initially with the dual combination, to be followed with the drug trio of simultaneous Gem + MK8776, followed by MK1775. To reduce the likelihood of toxicity, Gem doses lower than the typical “full” dose (100 mg/kg IP twice per week) were tested. We found that 25 mg/kg Gem twice in a day (8h apart), twice per week was not well tolerated even as a single agent, but both 25 and 50 mg/kg Gem once in a day, twice per week was tolerated when co-dosed with 25 mg/kg MK8776, and when this combination was followed 8 hours later by 60 mg/kg MK1775 (OG). A pharmacokinetic study revealed that plasma Gem (dFdC) concentrations were not altered by co-dosing Gem with MK8776, but intratumor dFdCTP (the active intracellular metabolite of Gem) was elevated at 1 – 8 hours after the 25 mg/kg Gem + MK8776 dose (AUC0 –last 49 +/- 7.9 hr.pmoles/mg tissue) compared to mice with 25 mg/kg Gem alone (AUC0-last 21 +/- 2.6 hr.pmoles/mg tissue). CHEK1 target inhibition was demonstrated in vivo, with abrogation of Gem-induced CHEK1 S296 autophosphorylation for at least 4h post Gem + MK8776 dosing, using Western blot analyses of tumor lysate. There was also increased γH2AX and pRPA32 S4/8 at 4 - 8 hours post-dose with Gem + MK8776 compared to Gem alone, indicating enhanced DNA damage. Pharmacodynamic and efficacy studies with the triple combination, will determine whether enhanced efficacy can be observed, when compared to full dose, single agent gemcitabine (100 mg/kg IP twice per week). Citation Format: Siang-Boon Koh, Frances M. Richards, Yann Wallez, Duncan I. Jodrell.{Authors}. Evaluation of scheduling for triple therapy gemcitabine/CHEK1 inhibitor/WEE1 inhibitor in pancreatic cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B62.

Abstract 3497: Mechanism-based scheduling of triple therapy gemcitabine/CHK1i/WEE1i in pancreatic cancer at submaximal yet synergistic concentrations

Sensitization of cancer cells to gemcitabine has been shown with checkpoint kinase CHK1 and WEE1 inhibitors. To rationalize and optimize the concomitant use of these three agents, we first performed growth inhibition assays on MIA PaCa-2 pancreatic cancer cells using gemcitabine in combination with either CHIR-124 (CHK1 inhibitor) or MK-1775 (WEE1 inhibitor). In silico analysis with three mathematical models (Bliss Independence, Loewe and Highest Single Agent) identified synergistic growth inhibition at submaximal (i.e. 8000 individual cells undergoing extensive fork collapse (compared to 0-1% with single agents) as evidenced by co-staining of yH2AX and hyper-phosphorylated RPA32. Conversely, submaximal gemcitabine+MK-1775 induced clear reduction of inhibitory CDK1 Y15 level compared to single agents. Consistent with this G2/M abrogation, in a Fucci-based CDT1/geminin-expressing MIA PaCa-2 stable cell line, gemcitabine+MK-1775 partially reversed the accumulation of the geminin-expressing (S/G2) population induced by gemcitabine alone. This was accompanied by an increase in the CDT1-expressing (G1) population, alongside aberrant mitotic cells with features of perturbed kinetochore-microtubule integrity. Based on these findings, we performed a series of long-term, kinetic live-cell imaging assays to determine the optimal scheduling for gemcitabine/CHK1i/WEE1i as a triplet at synergistic concentrations. In contrast to the widely proposed scheduling regimen, we found that delayed administration of CHK1i (at 24 hours), relative to gemcitabine, did not lead to synergy. However, concurrent administration yielded durable growth inhibition, even when the two agents were removed after 24 hours. This inhibition was further enhanced with the subsequent addition of WEE1i at 24 hours, but not continuation of a CHK1i. Together, our results illustrate differential mechanistic effects of CHK1 and WEE1 inhibitors with gemcitabine, when combined at sub-GI50 concentrations, and propose a novel triple agent schedule that will be evaluated in vivo. Citation Format: Siang-Boon Koh, Frances M. Richards, Esther Rodriguez, Scott K. Lyons, Duncan I. Jodrell. Mechanism-based scheduling of triple therapy gemcitabine/CHK1i/WEE1i in pancreatic cancer at submaximal yet synergistic concentrations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3497. doi:10.1158/1538-7445.AM2015-3497