Optogenetics in cancer drug discovery

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Expert Opinion on Drug Discovery

ISSN: 1746-0441 (Print) 1746-045X (Online) Journal homepage: http://www.tandfonline.com/loi/iedc20

Optogenetics in cancer drug discovery Michał Kiełbus, Jakub Czapiński, Adrian Odrzywolski, Grażyna Stasiak, Kamila Szymańska, Joanna Kałafut, Michał Kos, Krzysztof Giannopoulos, Andrzej Stepulak & Adolfo Rivero-Müller To cite this article: Michał Kiełbus, Jakub Czapiński, Adrian Odrzywolski, Grażyna Stasiak, Kamila Szymańska, Joanna Kałafut, Michał Kos, Krzysztof Giannopoulos, Andrzej Stepulak & Adolfo Rivero-Müller (2018): Optogenetics in cancer drug discovery, Expert Opinion on Drug Discovery To link to this article: https://doi.org/10.1080/17460441.2018.1437138

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EXPERT OPINION ON DRUG DISCOVERY, 2018 https://doi.org/10.1080/17460441.2018.1437138

REVIEW

Optogenetics in cancer drug discovery Michał Kiełbus Joanna Kałafut

a

, Jakub Czapiński a,b, Adrian Odrzywolski a, Grażyna Stasiak c, Kamila Szymańska a, , Michał Kos a, Krzysztof Giannopoulos c,d, Andrzej Stepulak a and Adolfo Rivero-Müller

a

a,e

a

Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland; bPostgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland; cDepartment of Experimental Haematooncology, Medical University of Lublin, Lublin, Poland; d Department of Hematology, St. John’s Cancer Center, Lublin, Poland; eCell Biology, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland ABSTRACT

KEYWORDS

Introduction: The discovery and domestication of biomolecules that respond to light has taken a light of its own, providing new molecular tools with incredible spatio-temporal resolution to manipulate cellular behavior. Areas covered: The authors herein analyze the current optogenetic tools in light of their current, and potential, uses in cancer drug discovery, biosafety and cancer biology. Expert opinion: The pipeline from drug discovery to the clinic is plagued with drawbacks, where most drugs fail in either efficacy or safety. These issues require the redesign of the pipeline and the development of more controllable/personalized therapies. Light is, aside from inexpensive, almost harmless if used appropriately, can be directed to single cells or organs with controllable penetration, and comes in a variety of wavelengths. Light-responsive systems can activate, inhibit or compensate cell signaling pathways or specific cellular events, allowing the specific control of the genome and epigenome, and modulate cell fate and transformation. These synthetic molecular tools have the potential to revolutionize drug discovery and cancer research.

Optogenetics; cancer; drug screening; drug targets; offtargets; cell motility; toxicity; drug resistance

1. Introduction Drug discovery requires the testing of thousands of molecules on cell assays and in vivo experiments, and thus it is imperative the fast measurements of cellular responses, target specificity, cellular stress and toxicity, and the detection of any possible off-target effects. While this is normally achieved through use of high-throughput and high-content screenings, using laboratory automation equipment and librariess of compounds and/or molecules e.g. siRNAs, there are approaching new technologies which can speed up drug and drug target discovery. Among these techniques is optogenetics – the measurement and stimulation of biological functions by photons. Unlike compounds, photons can be easily controlled in direction, time and space, making optogenetics an ideal system to precisely manipulate biochemical and cellular events with the highest flexibility and spatiotemporal precision [1]. Over the last decade, a series of naturally existing photoswitchable modules have been found in proteins in all kinds of organisms. These have been the basis of further synthetic systems which respond to varying wavelengths of light by stimulating biological processes [2–5]. Although optogenetics has been mainly linked to neurobiology, due to its origins using photoactivated ion channels and pumps, the field is slowly shifting to other areas of biomedicine. Here, we present a series of perspectives on how optogenetics can bring new

light to cancer drug discovery, as to date no other review of optogenetics in cancer drug development has been published. Although there are plenty of reports describing novel optogenetic tools, these mostly focus in their bioengineering and downstream control of cell signaling with relatively little vision to drug discovery. Thus, the need of a comprehensive overview on their potential on pharmaceutical discovery seems overdue. In Table 1, we summarize the light-responsive proteins and protein domains as well as categorize them as channels, receptors, catalyzers, dimerizers and actuators depending of their molecular function.

2. Cancer cell biology 2.1. Controlling intracellular signaling by channels and receptors Opsin receptors in microorganisms are in fact ion channels or ion pumps that become activated by light. By domesticating the Chlamydomonas’ channelrhodopsin (ChR2) [10,11] and archaeal halorhodopsin (NpHR) [12] to operate in mammalian neurons, the field of optogenetics came to light. These membrane ion channels allow the influx of Na+ (ChR2) or Cl− ions (NpHR), when stimulated by the correct wavelengths, which activates or inhibits, respectively, the membrane potential of neurons or myocytes.

CONTACT Adolfo Rivero-Müller [email protected] Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland; Cell Biology, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland © 2018 Informa UK Limited, trading as Taylor & Francis Group

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Article highlights ● ● ●

● ● ●

Optogenetic tools allow to reversibly manipulate cellular behavior with high precision Several existing optogenetic systems can be easily adopted for cancer drug discovery Drug target specificity and compensating mechanisms can be pinpointed by activation or inhibition of downstream signaling by lightresponsive actuators Light activating moieties allow experimental control of genomic and epigenomic states Prediction of toxicity and drug resistance mechanisms is simplified and multiplexed Optogenetic systems translate seamlessly to in vivo situations

Unlike their microbial counterparts, opsin receptors in mammalian cells are light-sensing membrane receptors which belong to the G protein-coupled receptor (GPCR) family and thus transduce intracellular pathway activation mainly via G proteins. In cancer cells, intracellular GPCR signaling is often dysregulated [13,14], thereby the ability to control single pathways is not only important to understand tumorigenesis, cell survival, immune evasion, angiogenesis, metastasis, proliferation, and drug resistance but also for drug discovery and toxicity. In order to create light-activatable GPCRs that trigger predisposed intracellular pathways, Airan and collaborators assumed that by replacing the intracellular loops, that’s where G proteins bind to, of the Gt-coupling green-absorbing rhodopsin receptor with those of the Gq-coupled human alpha-1-adrenogenic receptor (α1AR), or those of the Gscoupled beta-1-adrenogenic receptor (β2AR), would result in the light-induced activation of the desired G protein. Indeed, opto-α1AR activated adenylate cyclase and cyclic adenosine monophosphate (cAMP) synthesis upon red light illumination, while opto-β2AR effectively activated phospholipase C, resulted in an increase of inositol triphosphate (IP3) production after exposition to violet light [15]. These chimeric

receptors respond to light by activation of the same pathways that the native receptor from which the intracellular domain was taken (Figure 1(a)). Most GPCRs either induce the generation of cAMP or its destruction – via activation of phosphodiesterases. Yet, GPCRs can also trigger other downstream signaling cascades such as the IP3-dependent signaling pathway that plays roles in cell survival and apoptosis and thus related to many types of malignancies [16]; the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) involved in survival and proliferation; the small GTP-binding proteins (Ras and Rho family) that are main players in cell migration; and the activation of a plethora of downstream transcription factors. These signaling cascades can also be activated by, and often are mostly associated to, tyrosine receptors (growth factor receptors), cytokine receptors, and integrin signaling. To dissect the contribution of each of these pathways to cancer biology, they need to be regulated separately, and here is where optogenetics actuators shine.

2.2. Intracellular pathway activation by optogenetic catalyzers, dimerizers and actuators The success of optogenetic opsins in animal models, has triggered an extensive search for other light-responsive proteins in all kingdoms of life. To date, there is a series of discovered or engineered proteins which can catalyze biochemical reactions upon light activation. These can bypass the membrane receptor level and activate single downstream signaling pathways. Among these proteins is bPAC, a photoactivatable adenylyl cyclase from Beggiatoa sp. that generates cAMP upon illumination [65] (Figure 1(b)). Cyclic AMP plays a variety of biological roles and has been linked to cancer cell survival, inhibition of apoptosis, inhibition of cancer cell migration, as well as to confer resistance to antineoplastic drugs, in a context- and cell-dependent manner [17–20]. bPAC is yet to be used in cancer research, but it has been used to determine the decisive role of cAMP in neurotransmission, in contrast to

Table 1. The light-responsive proteins and protein domains. Light-responsive modules/proteins Dimerizers CRY2 (Blue-light receptor cryptochrome 2) CIB1 (Cryptochrome-interacting basic helix-loop-helix 1) PhyB (Phytochrome B) PIF (Phytochrome interacting factor) CRY2 (Blue-light receptor cryptochrome 2) CRY2a (Blue-light receptor cryptochrome 2) pMag (Positive magnet) nMag (Negative magnet) Actuators Dronpa (Photoactivatable green fluorescent protein) LOV (Light/Oxygen/Voltage domain) Channels ChR2 (Channelrhodopsin 2) NpHR (Halorhodopsin chloride pump) Rhodopsin bPAC (Photoactivatable adenylyl cyclase) LAPD (Red-light-activating phosphodiesterase) a

Receptors Catalyzers

Activation wavelength (nm)

Ref.

Blue light (466) Red light (∼650–700) Blue light (460–480) Blue light (470 ± 20)

[6] [7] [8] [64]

~500 – dark monomer ~400 – green tetramer Blue light ~450

[32,37] [9,35]

Blue light (~470) Yellow light (~589)

[10] [12]

Green-blue light (504 ± 6)

[15]

Violet light (405) Far-red light (~700)

[22] [27]

CRY2 can undergo homo-oligomerization in the lit state. The table presents all the most commonly used proteins or domains that respond to light. They have been classified based on their action upon photostimulation into dimerizers, actuators, channels, receptors and catalyzers. For the first, their binding partners are also described. The activation wavelength for each system is presented.

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Figure 1. Optogenetic control of GPCRs and second messengers. (a) Photoactivatable GPCRs; (b) Photoactivatable adenylyl cyclase bPAC; (c) Light-activatable phosphodiesterase (PDtE2A).

ChR2 photodepolarization, in the nematode Caenorhabditis elegans [21]. bPAC has also been used to rescue cAMP signaling in cells where the endogenous soluble adenylyl cyclase has been knocked out e.g. mice sperm which recovered their motility and fertilization potential upon photoactivation by blue (405 nm) light [22]. In cells, cAMP is rapidly hydrolyzed by phosphodiesterases (PDs) which are commonly activated downstream several GPCRs. PDs have been found overexpressed in several tumors, and thus have been suggested as potential drug targets [23,24]. Moreover, overexpressing PD-4 enhances the effects of hypoxia and downstream gene activation by hypoxia inducible factor 1 (HIF-1); a factor associated with metastasis, angiogenesis, and therapy resistance [25], leading to increased lung cancer cell proliferation and colony formation [26]. With this in mind, a light-activatable phosphodiesterase (PDtE2A) has been bioengineered by fusing dimeric human phosphodiesterase 2A with the photoactivatable PhyB, a protein which changes its conformation upon red light irradiation, the resulting chimeric enzyme responds to red-light by hydrolyzing cGMP and cAMP [27] (Figure 1(c)). Thus, the regulation of intracellular cAMP by optogenetic catalyzers allows for the careful scrutiny of the molecular events which lead to cellular alterations and cancer progression. Many of the biological roles of cAMP are the result of cAMP-dependent protein kinase (PKA) activation which in turn phosphorylates a number of metabolic enzymes and transcription factors to name but a few, depending on PKA’s localization. PKA has been considered as a cancer biomarker as well as a potential molecular target for cancer therapy [28]. In order to create a light-activatable PKA that works independently of cAMP and at specific intracellular locations, O’Banion

and collaborators used PKA constitutively active mutants, with low phosphotransferase activity in the cytoplasm, that when targeted to specific intracellular sites, using a fusion of PKACRY2 and organelle-located CIB1, these optoPKA mutants concentrate and phosphorylate a colocalizing reporter protein [29]. The authors suggest that other opto-kinases could be generated using similar architectures as the mutations used for optoPKA reside in conserved residues in other protein kinases. Tumor progression is caused by an elevated proliferation potential, the acquiring of a migratory phenotype and an increased survival of cancer cells. These features are regulated mostly by MAPK/ERK-, JUN-, and/or p38-dependent signaling pathways [30], as well as by the ‘guardian of the genome’ – the p53 protein. Thus, any abnormality in cell signaling that affects the regulation of cell division becomes a potential target of cancer therapy. Often, these signaling cascades are coactivated due to common upstream mechanisms. Growth factor receptors, some GPCRs, cytokine receptors and stress can activate, via G proteins (e.g. Cdc42, Ras or Rac), MAPKKKKs (MAP kinase kinase kinase kinases) such as HPKs (Histidine protein kinases), PAKs (p21 activated protein kinases) and GCKs (Germinal center kinases), which in turn activate MAP3 kinases (Raf1/ASK1, MLK, MEKK). Each of these activates MEK1/ 2, MKK4/7 or MKK3/6, respectively. MEK1/2 activates ERK1/2, MKK4 phosphorylates JNK and, probably, p38, while MKK3/6 activates p38 [31]. The overlapping and diversifying nature of these signaling pathways make them hard to study separately. Fortunately, there is a palette of novel optogenetic tools that allow the selective manipulation of single players. Opto-activation of the MAPK/ERK signaling pathway, which results in an increase in proliferation, cell survival, and

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development of cancer cells, has been achieved by the use of several photoactivatable tools in living cells: The first module (Figure 2(a)) exploits the PhyB/PIF opto-dimerizers (see Table 1), having on one hand PhyB localized to the cell membrane (where also RAS kinase is normally situated) and on the other a cytoplasmic PIF fused with a constitutively active SOS protein (actSOS). Red-light irradiation relocalizes PIF-actSOS to the membrane where actSOS activates RAS, triggering MAPK and downstream ERK activation [67]. The second set of tools to photo-control MAPK/ERK is the photo-stimulated kinases psRaf1, psMEK1 (shown in Figure 1(e)) and psMEK2. These tools work by flanking the respective kinase with two Dronpa domains: in dark state, Dronpa domains bind to one another, inactivating the kinase by blocking the active site. Upon light stimulation, the Dronpa domains dissociate undocking the kinase active sites (Figure 2(b)). These tools have been considered as all-optical cell-based assay for screening inhibitors, for example they have allowed to uncover the direct rapid inhibitory feedback loop from ERK to MEK1 in mammalian cells as well as in the nematode C. elegans [32]. An additional tool for MEK-ERK photoactivation has been created using a set of engineered CRY2 variants with different (high or low) homo-oligomerization abilities, fused with Raf1 protein – whose function is dimerization dependent. Upon blue-light irradiation, Raf1-CRY2high shows rapid and extensive clustering, producing higher level of ERK activation, whilst CRY2low-Raf1 only produces a slight increase in activation of ERK in HEK293 cells [33]. The JNK kinase family regulates many cellular features including inflammatory responses, morphogenesis, proliferation, differentiation, survival, and cell death. Moreover, persistent JNKs activation is involved in the development and progression of different types of cancer. Therefore, JNK kinases are also considered attractive targets for therapeutic intervention, mainly with the use of small kinase inhibitors [34]. In order to photo-command this pathway, the group to Michael Courtney created a light-sensitive JNK inhibitor (OptoJNKi, Figure 2(c)) using the clam shell-like LOV (Light-Oxygen-

Voltage) domain – LOV docks on its Jα domain in dark-state, but undocks upon blue light stimulation. By inserting the N-terminal fragment of a constitutive inhibitor of JNK [MAPK8IP1, mitogen-activated protein kinase 8 interacting protein 1, a.k.a. JNK-binding domain (JBD)], adjacent to the Jα domain, LOV shields it for interacting with any other protein. In the lit-state, LOV uncages OptoJNKi which immediately binds to and inhibits JNK as well as downstream phosphorylation of c-Jun (Figure 2(c)) [35]. An important and unexpected finding is that the frequency of inhibition of JNK can resonate on its downstream effects – the precise periodicity has a larger effect than shorter or longer illumination periods. Although their experiments were performed in neurons, the effects might have implications in other cell types, suggesting that therapeutic use of JNK inhibitors shall be adjusted to the cellular context to ensure maximal JNK inhibition and downstream resonance [35]. In addition, the same group developed an optogenetic inhibitor of p38 MAPKs, kinases implicated in adaptive responses to cellular stresses such as inflammatory cytokines, osmotic or heat shock, and ultraviolet radiation by regulation of apoptosis, cell proliferation, autophagy, and cell fate. Over-activation of p38 MAPKs is usually associated with the malignant phenotype and poor patients’ prognosis of multiple tumor types. p38 MAPKs play multiple, and often contradictory, roles e.g. as regulators of cell death, where p38 kinases play prosurvival or proapoptotic roles in a context-, isoform- and/or cell typedependent manner [35]. Moreover, p38 MAPKs modulate cell migration, drug resistance, and invasive potential, thus an intensively search for p38 inhibitors is underway [36]. The optogenetic p38 inhibitor (Opto-p38i) was developed using the same approach as in OptoJNKi (Figure 2(d)), an inhibitory peptide was masked by the LOV domain in dark state. In this case, the short inhibitory peptide was taken from MKK3’s D-domain, a selective inhibitor of p38 kinase. The use of OptoJNKi provides for the first time evidence for resonance in JNK-signaling circuits [35], which is at odds with traditional kinase cascade models established with the use canonical methods.

Figure 2. Optogenetic tools to manipulate intracellular signaling pathways. (a) Activation of Ras downstream signaling using opto-SOS system; (b) MAPK/ERK signaling pathway stimulated by photoactivatable MEK1 kinase; (c-d) Activation of JNK or p38 kinase by light; (e) Inactivation of nuclear p53 using a LOV-caged nuclear export signal (NES), photo-undocking exposes NES to CRM1, who assists its export out of the nucleus; (f) Photo control of a transcriptional activator (TA) by light activation and elevated calcium concentration (FLARE) where light undocks the recognition site of a protease from LOV and calcium activates calmodulin (CaM)-protease recruitment to calmodulin binding protein (CBP), releasing the TA; (g) Unlike FLARE, iTango is based on the recruitment of β-arrestin, fused with a protease fragment, to an activated GPCR, this is insufficient to cleave the TA, yet upon illumination recruitment of CRY2 fused with the complementing fragment of the protease is also recruited to the GPCR-arrestin complex and the protease recognition site is exposed from LOV. This system requires the activation of the GPCR by its ligand + light.

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p53 is a nuclear transcription factor that is considered one of the canonical tumor suppressors as more than a half of human cancers carry deleterious TP53 mutations that act as dominant-negative inhibitors toward wild-type (WT) p53. WT p53 prevents the propagation of cells suffering DNA damage primarily through the transactivation of its target genes, these are involved in numerous cellular processes such as cell cycle arrest, DNA repair, metabolism and/or apoptosis. In response to cellular stresses, e.g. DNA damage, p53 accumulates in the cell nucleus. In order to control p53 nuclear functions, a p53 light-inducible nuclear export system (LEXY) (Figure 2(e)) has been generated. The principle of LEXY rests on the use of LOV2 caging a nuclear export signal (NES) at one of the termini of p53 protein. Upon light illumination, the NES peptide is exposed to interact with the major mammalian export protein CRM1 (Chromosomal Maintenance 1), which facilitates the export of the Opto-p53 out of the nucleus. To prove its functionality, opto-p53 was expressed in TP53 knock-out cells, which regained p53 activity in darkness. Upon light exposure, LOV2 uncaged the NES, followed by ejection of p53 to the cytoplasm and the loss of p53 transcriptional activity – measured as a decrease in the levels of one of its target genes, p21 protein [37]. We could reason that detailed studies regarding the complex cytoplasmic roles of p53 will be performed using this elegant system. In fact, cytoplasmic p53 is involved in triggering apoptosis, a fine-tuned balance between the levels of cytoplasmic p53 and those of interacting proteins, such as Bcl-xL and Bcl-2, both inhibitors of p53-induced apoptosis [38]. The inverted system, where a nuclear localization signal (NLS) is controlled by LOV also exists [39], enabling accumulation in the nucleus in lit state, although has yet to be reported for p53. Thanks to the advances in gene editing technologies, the deletion of the endogenous TP53, or better its replacement (knock-in) by opto-mutants, can be nowadays achieved. Another signaling protein which has been effectively controlled by light, is Cyclin-dependent kinase 5 (CDK5), whose upregulation correlates with increased malignant progression, migration, and invasion in different cancer types [40,41]. Photoactivatable-CDK5 (psCDK5) was created as cell-based assay for screening inhibitors, following a similar approach as that described for psMEK1 and psMEK2 kinases using two flanking Dronpa sub-domains, in this case encaging the CDK5 functional core in dark state. Upon light stimulation, psCDK5 rescued the phenotype of Cyclin Y-knockout neurons of worms [32]. Cell signaling often culminates with the activation of transcriptional regulators, such as transcription factors and/or transcriptional repressors. In an attempt to control transcriptional regulators using light, Wang and coauthors recently developed a system named fast light- and activity-regulated expression (FLARE). Although this tool was invented to be used in neurobiology, FLARE system allows to conditionally upregulate target gene expression upon light activation plus cytoplasmic calcium (a common second messenger upon a myriad of cell communication events). FLARE is a 2-step system, where one component is a membrane-anchored calmodulinbinding peptide fused with a LOV domain encaging a TEV protease cleavage site, followed by a transcriptional activator (TA). The second component is a chimeric fusion of calmodulin

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(CaM) and TEV protease. Light activation of LOV results in uncaging the TEV recognition site. Yet, to function, Ca2+ is required to activate CaM, which then binds to the calmodulinbinding peptide, bringing TEV protease to the complex and releasing the TA to translocate into the nucleus and activate downstream gene expression [42] (Figure 2(f)). This system requires a coactivator besides light, which can be molecular event triggered by an activated receptor as far as intracellular calcium release occurs. In the same vein, an elegant optogenetic system that can engage virtually any GPCR as coactivator was created by Lee and collaborators – iTango [43]. This tool requires the activation of a GPCR by its ligand (agonist) plus light to become activated. iTango is based on 3 synthetic proteins: (1) a GPCR e.g. dopamine 2 receptor (DRD2), with a long C-terminus from V2 vasopressin receptor (β-arrestin2 binding partner), CIBN (a shorter form of CIB1), a LOV2 domain shielding a TEV cleavage site, and a transcriptional activator (TA); (2) β-arrestin2 protein fused to the N-moiety of TEV protease (TEV-N); and (3) CRY2 protein fused to the complementing C-terminus of TEV (TEV-C). Ligand activation of the GPCR causes β-arrestin2-TEV-N recruitment but not TEV recognition site cleavage. Upon blue light illumination CRY2 dimerizes with CIBN, joining the complementing moieties of TEV, and LOV2 simultaneous uncovers the TEV cleavage site – resulting in the proteolytic release of the TA. As most new optogenetic actuators, iTango has been used in vivo to study the activation of dopamine neurons in the brains of mice [43]. The main reason for this is that in neurobiology the readouts are easily determined. Both systems will probably be adapted to cancer drug discovery as both, GPCR and calcium signaling, are involve in tumorigenesis and/or processes important in invasion and metastasis, as well as in apoptosis regulation. The disadvantages of these systems are that they require multiple modules, each expressing at different levels, the readout is the use of a reporter gene expression which is rather slow and not fully quantitative, and that endogenous signaling events might affect their functionality and noise-to-signal levels. Finally, cancer metabolism is an area where no direct optogenetic system yet exists, but that could change with the discovery of a light-activatable enzyme which catalyzes lipid conversion to hydrocarbons [44], although this remains to be experimentally tested in mammalian cells. The control of metabolism may be achieved by indirect means however, such as ion channels, as has been demonstrated by disrupting of the synchronization of pancreatic beta cells transfected with the NpHR Cl− pump, which affects their coordinated response to glucose and secretion of insulin [45].

2.3. Optical control of the genome and epigenome Cancer not only arises and progresses by mutagenesis but through changes in gene and epigenetic regulation. Yet, controlling specific endogenous gene regulation has been traditionally troublesome, due to the complexity of the genome, and thus mostly performed as silencing genes before translation (siRNAs and miRNAs), while changes in epigenetic modifications was indiscriminately performed by inhibition of

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epigenetic editors such as histone acetylases (HATs) or deacetylases (HDACs). Several HDAC inhibitors (HDACs) (e.g. valproate, romidepsin, and suberanilohydroxamic acid – SAHA) as well some de novo methyltransferase inhibitors (DNMTis) (e.g. azacitidine and decitabine), collectively called epi-inhibitors, have been approved in the clinic for the treatment of hematological malignancies, or are being tested in clinical trials against many solid (e.g. melanoma, cervical, lung and breast) tumors. The mechanism of action of epi-inhibitors has been assumed as the reactivation of silenced genes by loss of epigenetic marks (histone modifications or DNA methylation). Indeed, evidence exists that re-expression of some genes results in a more sensitive phenotype to other treatment modalities such as chemo or hormonal therapy. To exemplify this, the demethylation of the promoter of the estrogen receptor alpha (ESR1), resulting in de novo expression of this nuclear receptor, sensitized hormone-irresponsive breast cancer cells to tamoxifen [46]. Yet, HDACs indiscriminatingly affect proteins other than histones, which might cause distinct phenotypical effects, e.g. estrogen receptor-alpha (ERα)-positive cells treated with SAHA responded by decreasing the expression of ERα, due to the increased acetylation of proteins that stabilize ERα such as heat-shock protein 90 [47]. Since epi-inhibitors lack selectivity, generate genome-wide perturbation [48], and disrupt cell signaling pathways that might, in some cases, result in unforeseen effects e.g. activation of epithelial-mesenchymal transition and metastatic factors [49,50], epigenetic reprogramming should be performed by targeting specific genes or regions and their downstream effects in cellular phenotypical changes and responsiveness to drugs [51]. And the best way to do this is to cause these changes suddenly to avoid compensatory mechanisms and other long-term effects. The advent of gene editing technologies, notably Transcription Activator-Like Effectors (TALEs) and Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR/ dCas9 – a nuclease-dead Cas9 that maintains its DNA-binding abilities), has made possible to manipulate genetic and epigenetic information at specific genomic loci. Both TALEs and CRISPR/dCas9 function as sequence-specific DNA-binding domains (DBDs), and by attaching gene regulators (activators or repressors of transcription) or epigenetic editors such as DNA and histone modifying enzymes, it has become possible to control single genes or chromatin regions [52]. These systems have further been adapted for optogenetic manipulation, by fusing an optogenetic dimerizer (either CRY2 or PhyB) to a DBD (TALE or dCas9), the DBD binds innocuously to the target sequence, a promoter. The dimerizer partner (CIB1 or PIB, respectively) has been fused to either a transcriptional activator, e.g. VP64, a transcriptional repressor, or an epigenetic modulator such as p300 or KRAB, from here onwards generalized as ‘editors’. In addition, a split dCas9 has also been created so it is reconstituted when the halves are brought together by pMag and nMag photo-dimerizers (each fused with a different half of dCas9). In all cases, photoinduced dimerization brings the corresponding effector to the genomic/chromatin region of interest where it acts by activating or inhibiting transcription, or modifying chromatin structure [53–57] (Figure 3(a–b)). Optogenetic regulation of site-

Figure 3. Optogenetic regulation of the genome: (a-b) Dimerization triggered by light allows to upregulate (TA, transactivator) or knock-down (TR, transcriptional repressor) specific loci targeted using dCas9 or TALEs as DNA binding domains; (c) Opto-Cas9 and opto-CRE are gene editing modules that become functional upon dimerization of photo-activatable proteins, and are able to excise a gene upon activation.

specific subtelomeric DNA-methylation was achieved by Choudhury and coworkers, where they used CIB1 fused to telomere repeat binding factor-1 (TRF1) and CRY2 fused with DNA methyltransferase3A (DNMT3A). Increased methylation level at sub-telomeric CpG sites after blue-light activation resulted in progressive increase in telomere length over three generations of HeLa cells [59]. The same group has been able to optogenetically regulate the methylation status of proneuronal Ascl1 (Mash1) gene promoter in neural stem cells (NSCs). Here, CRY2 was fused to the catalytic domain (CD) of either DNA-methyltransferase3A (DNMT3A-CD) or demethylase ten-eleven dioxygenase-1 (TET1-CD); while CIB1 was fused to a TALE designed to bind to the Ascl1 promoter. This system selectively modulated the methylation state of Ascl1 promoter, what resulted in Ascl1 expression upon light activation in neural stem cells. Hypomethylation of Ascl1 promoter by TET1-CD resulted in Ascl1 expression, which caused neuron generation in rat dorsal root ganglion NSCs, whereas Ascl1 promoter hypermethylation produced by DNMT3A-CD resulted in inhibition of Ascl1 expression, what induced glia generation in striatal NSCs [60]. Although, these examples of optogenetical modifications and their phenotypical results are not strictly related to cancer or drug discovery, we can predict that these systems will soon be applied in cancer drug discovery since all these processes play important roles in cancer cell immortalization and drug responsiveness. The control of epigenetic marks has already provided valuable lessons. Two examples: first, highly methylated promoters have low affinity for transcription factors and the genes under such promoters are not actively transcribed [61]; second, by directing epigenetic control of specific genes it is possible to induce cell differentiation e.g. mouse fibroblasts into myoblasts or inactive to active neurons [62]. The main advantage of light-inducible systems is that the control of endogenous genes can be fine-tuned. For example, a directly proportional expression of TP53 to the dose of illumination was achieved by using dCas9-CIBN and CRY2p65 targeting the promoter of TP53 in two bladder cancer cell lines [63]. This will allow dissecting the functions of

EXPERT OPINION ON DRUG DISCOVERY

many signaling proteins that have been found having antagonistic effects depending of their level of expression or activation, and such can now be performed in vitro as well as in vivo thanks to optogenetic modules.

2.4. Genomic modifications Gene deletion or activation (by e.g. inversion) has been traditionally achieved by the use of recombinases, notoriously CRE recombinase, although one should note that gene editing techniques such as CRISPR/Cas9 have recently entered onto the scene due to their programmable specificity. Both, CRE and Cas9, have been split in half, where each half is fused to an opto-dimerizer (CRY2/CIB1 or pMag/nMag, respectively). Exposing cells to the correct wavelength induces complementation and functional enzyme formation [64] (Figure 3(b–c)). There are 2 advantages of Cas9 to CRE, first the DNA target for Cas9 can be an unmodified endogenous gene, and second dCas9 can be used as DBD carrying epigenetic editors [64]. Recombinases such as CRE and FLP, however, are able to delete or invert genomic regions, or even create chromosomal translocations, with incredible accuracy and efficiency, which is something that Cas9 is unable to perform. Nevertheless, an endogenous gene or genomic region must be modified beforehand by insertion of two recombinase’s recognition sequences (loxP or Frt sites for CRE and FLP, respectively) flanking a region to be deleted or inverted. Opto-CRE has already been applied to control gene activation in specific neurons in the brain of a murine model [66], and one could easily envision similar experimental settings in cancer biology e.g. the deletion or activation of oncogenic or drug-resistance factors. Genomic rearrangements are common in cancer, in some cases it involves the deletion of one or several tumor suppressors, in others the formation of gene fusion with novel properties e.g. Philadelphia chromosome during large-scale complex genomic rearrangements (chromothripsis, breakagefusion-bridges, and double minutes), and yet in other cases changes in chromatic structure resulting in differential expression of the genes in the affected locus [66,68]. When it comes to gene ‘deletion,’ this could be done by permanently knocking out the gene of interest or to

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temporarily knock it down by using any of the light controllable transcriptional regulators mentioned above. The latter is similar to what is already in practice in search for target genes by means of silencing RNAs. The advantage of the light-inducible systems is that they do not require additional transfections or viral transductions, they can be stably expressing the opto-system without any additional effect until lit, and also these cells can be co-cultured with other unmodified cell lines, control non-cancerous cells or supporting cells, or even in mini tissues and 3D cultures where transfection is troublesome, allowing drug testing of target and control cells in the same well [69]. In practice, a drug-sensitive cancer cell line is optogenetically triggered for a genomic alteration (mutation, deletion, inversion, or gene fusion), and drug testing is performed again to study the mechanisms of drug resistance due to single genetic events. Such assays will provide two main readings, first the predictability of potential drug resistance, for which alternative treatments could be planned; and second, the testing of compounds acting on direct and compensatory pathways.

2.5. Controlling cell migration Migration, probably the most alarming process in oncology, is optogenetically feasible by a variety of elegant methods. Two of these tools are based in the polymerization of the cytoskeleton by using a constitutively active Rac1 (*Rac1) – a regulator of actin cytoskeleton and thus migration. In the first case, a photoregulated LOV-lock conceals the interacting domain of *Rac1 for its effector protein [70], while in the second a cell membrane-anchored PhyB recruits PIF fusion with *Rac1 [71] (Figure 4(a–b)). In a slightly different twist, a caged ITSN2 protein, a master regulator of the actin cytoskeleton Rho family GTPases, sandwiched between two Dronpa monomers, one of which was tagged to the membrane by the CaaX motif, becomes uncaged upon cyan light [72,73]. The result in all cases was the formation of filopodia at the cells edges producing cell movement of the direction of light of the correct wavelength (458, 650, and 405 nm, respectively) (Figure 4 (a–b)).

Figure 4. Control of cell motility by light. (a-b) Cytoskeleton remodeling and cell polarization stimulated by light via constitutively active Rac1 (*Rac1) protein, where the interaction domain to Rac1 effector is blocked by LOV in dark. The formation of actin filaments on one edge of the cell triggers forward migration. (c) Photoactivatable CXCR4 (PA-CXCR4), a chimera of the rhodopsin and the CXCR4 receptors, induces cell polarization in the direction of the light source.

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Cell directional migration has also been achieved by modifying the CXCR4 chemokine receptor, whose role is to guide cell migration toward a chemokine concentration gradient (Figure 4(c)). The chimeric rhodopsin-CXCR4 responds to 505 nm light activation similar to that of a chemokine gradient of the naïve CXCR4, and thus T cells which express the PACXCR4 receptor become polarized and follow a gradient of light [74], which has been used in opto-immunology, a subject out of the scope of this work and thus we only refer the reader to specialized reviews in the topic [75,76]. Migration assays are commonly used in drug discovery, as drugs that inhibit migration are potential anti-metastatic agents. Yet, many of the scratch/wound assays do not well correlate to cell migration, partly due to the lack of directionality of cells to migrate and partly through other technical issues such as cell and matrix damage during the removal of cells [77,78]. One of the advantages of optogenetic-driven migration is that it is directional, cells migrate in the direction of light, and the origin of migration can be perfectly defined, thus the quantification of migration will be more reliable and accurate than current systems. Moreover, most transmigration assays involve chambers separated by a porous membrane, where one chamber is loaded with a chemoattractant that forms a nonlinear gradient, adding an additional factor in the assay that can hardly be controlled [79]. Since optogenetic systems are various and based in different molecular players, they can be used as orthologous migration assays to pinpoint the mechanism of action of a small molecule. Moreover, cells having such systems could be optogenetically labeled (e.g. fluorescent protein) and co-cultured with unmodified cells of the same type or different type i.e. fibroblasts, for easy quantification. Additionally, migration assays could be complemented with some of the other opto-signaling regulators, as several of these biological processes are linked. For example, the MAPK/ERK pathway is known to be involved in cell migration [30] and the JNK pathway converges with other pathways of cell migration as well [80].

3. Light to the rescue: testing toxicity Most phase II and III clinical trials fail at the efficacy (52%) and safety (24%) of the drug [58]. The toxicity of drugs is responsible for the withdrawal of approximately one-third of potential drugs, and significantly contributes to the high cost of the development of new drugs, especially when its detection occurs during advanced clinical trials, or at the introduction of large-scale drug production [81]. Therefore, novel systems which can determine the toxicity and specificity of drugs at early stages, are urgently needed. Moreover, tumor cells often evolve drug resistance for which there is virtually no standardized assay. Systems based on optogenetic readouts have been used in cancer drug discovery for many years. Best known examples are the use of Nuclear Factor kappa B (NFkB)-sensor: where a reporter protein such as GFP or luciferase is placed under the control

of a minimal promoter that contains multiple tandem repeats of the NFkB transcription-responsive element (TRE), this biosensor allows quantitatively determination of NFkB activity [82]. NFkB is a heterodimeric protein with transcription factor-like properties, which is the downstream response of many growth factors, proinflammatory cytokines, oxidative stress, hormones, DNAdamaging drugs and viral infection [83] and it is a central regulator of stress responses as well as a blocker of apoptosis in several types of cancer cells, which is an important marker to neoplasias and drug resistance. Nevertheless, the rapid but accurate assessment of drug toxicity requires the development of new high-throughput techniques to test a large number of substances at experimental level. Among these techniques, we can include optogenetic proteins such as ion channels: channelrhodopsin 2 (ChR2), halorhodopsin (NpHR), CheRiff, voltage-sensitive fluorescence proteins such as VSFP2.3; which have been originally domesticated to control and monitor neuron physiology and muscle contraction (skeletal as well as cardiac) in several in vitro and in vivo studies [84–93]. These are some of the most sensible, and fast reacting, cell types to environmental changes and damage, and thus excellent reporters of cell stress and toxicity. In light of this, the group led by Joel M. Kralj adopted a strategy to assess cardiotoxicity screening by simultaneous optogenetic pacing followed by voltage and calcium imaging [92]. Pacing was achieved through the use of a freshwater algae channelrhodopsin variant CheRiff, while voltage and Ca2+ imaging were visualized via CaViar, a double reporter system having a voltage-sensing red fluorescent protein, QuasAr2, and a blue Ca2+-indicating protein, GCaMP6f [92]. In this manner, cell responses can be monitored by either microscopies, or by a fully automated platform such as OptoDyCE. The latter platform, pioneered by the group led by Emilia Entcheva, allows the control of all dynamic electrophysiological functions of a single heart cell by a simultaneous optogenetic excitation of ChR2, and optical sensing of voltage and calcium levels by red-shifted dyes [94]. Integrating optical pacing with optogenetic probes is crucial to determine drugs’ cardiotoxicity and arrhythmia predictions [94]. Drugs affecting cell function will immediately show abnormal cell contraction or firing, which could be observed in real time – and both stimulation and readout is performed optogenetically. Rescuing experiments where the function of ion channel is compensated by a photoactivatable ion channel can be used to determine the mechanism of action of a neuro/cardiotoxic drug, and in some cases might help to find chemoprotectants that compete for binding to the ion channel. Ion channel inhibition can increases the risk of arrhythmia which may lead to sudden cardiac death – the drug-induced form of long QT syndrome (LQTS) [95]. For example, inhibition of hERG (Human ether-a-go-go-related) potassium ion channel, an essential player in the normal electrical activity of the heart, has been the main cause for the withdrawal of several drugs in late stage clinical trials [96], and therefore hERG inhibition should be constantly analyzed during drug development [95,97–99]. Photo-gating potassium channels have been created by using photo-liable inhibitors that in dark state block the K+

EXPERT OPINION ON DRUG DISCOVERY

channel, but upon 500 nm light reestablish the transport of K+ out of the cell [100]. These systems require the covalent binding of a chemically modified ligand to a modified channel [101], but since some K+ channels work as dimers (like TREK1 potassium channel), Sandoz and collaborators rationalize that one mutant subunit that is unable to reach the cell membrane, can be rescued by heterodimerization to the endogenous second subunit, resulting in a heterodimer that can be photo-modulated to control neuronal action potential [100,102]. Another optogenetic tool, which could be used for cardiac toxicology (e.g. a spatiotemporal and reversible control) is blue-light-induced K+ channel 1 (BLINK1). BLINK1 was developed by fusing LOV2-Jα module to the small viral potassium channel Kcv. The BLINK1 system has been proved in vitro and in vivo, in the latter case it was used to reversibly inhibit the escape response of light-exposed zebrafish larvae [103]. Stem cells (SCs) and induced pluripotent SCs (iPSCs) are becoming an integral part of the drug discovery toolbox, because they can be differentiated into any cell type [104] which is ideal to study drug effects in many cell types as it occurs in physiological conditions. Induced cell differentiation is troublesome due to the necessity for special culture conditions for cells to differentiate. Although SCs can generate any cell type, their ability to differentiate into functional differentiated cells has not been fully demonstrated. However, there are established ways to differentiate SCs into hepatic, cardiac, pancreatic and neuronal types. For other cell types, the differentiation approaches are yet to be standardized. Current differentiation strategies involve stimulation by growth factors, but these are inefficient and experiments are poorly reproducible, moreover they often require an extensive number of differentiation steps that result in a heterogeneous population. Many of these conditions are expensive and take several days to take place [105]. How optogenetics can surmount these obstacles? The answer lies within the very systems that we have described above, the control at the genetic or signaling levels of key molecular players of differentiation. This concept has already been applied to the differentiation of SCs to neural progenitors by optical expression of the neural differentiator Brn2 (Brain2) [106], using a transgene carrying Brn2 under a promoter having a recognition sequence for GAVPO, a Gal4-VVD-p65 chimeric protein, who upon blue light activation homodimerises and binds to its DNA recognition site. Brn2 is a competitor of Oct4 for binding to Sox2. Oct4, Sox2, and Nanog form a self-regulatory network that maintain cell stemness. Thus, high expression of Brn2 displaces Oct4 from Sox2 to induce expression of neuronal differentiation genes [106,107].

4. Drug resistance and tumor relapse Far too often, patients’ tumors relapse after primary neoplasias have been successfully treated. The new neoplasms usually develop from cancer stem-like cells. Cancer stem cells (CSC) are responsible for, among other things, the ‘production’ of various types of cancer cells within the tumor (asymmetric cell division) especially in early stages of tumor development [108], an evolved resistance to chemo- and radio-therapies [109]. In addition, CSC can be dormant for decades [110].

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Finding CSCs targeting drugs is urgently needed, which strongly emphasizes routinely tests on CSCs in the drug discovery pipeline. Stem cell factors (especially Yamanaka’s four: OCT4, SOX2, Klf4, and c-Myc), used to produce induced pluripotent stem cells (iPSCs) [111], have been also used to convert non-tumorigenic cells, e.g. MCF-10A, fibroblasts or glioma cells, into induced stem-like cancer cells (iCSC). These iCSCs fulfilled all the conditions necessary to recognize them as CSCs: expression of stem cell markers [112], generation of tumors composed of different cell lineages after implantation in nude mice, and resistance to chemotherapeutics [113–116]. Optogenetical systems can regulate Sox2, OCT4, Klf4, and c-Myc expression (e.g. based on opto-DBDs) allowing to control the transformation of differentiated cells into iCSC, or to block the stemness of CSCs by knocking-down these factors. Precise and reversible control of cell differentiation may have many practical applications in anti-cancer drugs development: first, it allows detailed studies on the tumor formation process, as well as the mechanisms for overcoming of chemo-resistance. Yet, a poorly explored field is the functional analysis of downstream genes regulated by these factors to determine those involved in drug resistance – something that can be performed by e.g. opto-CRISPR-interference using libraries of gRNAs.

5. A dark side of light? The use of optogenetic systems may cause cellular and/or environmental responses, a subject recently discussed by Allen and coauthors [117]. Nevertheless, we need to break the possible side effects down into: the effects of exogenous DNA delivery to cells and the effects of its expression; light physical influence on the cells; direct voltage driven effects; specific ions influence on signaling molecules or cell network/ environmental side effects. Transfecting or transducing cells with optogenetic modules, as with any transgene, might adversely affect the cells’ behavior resulting in cell death or immune responses against transfected cells if these are introduced into an organism [118,119]. Light, when absorbed by cells or tissues, results in heat. As temperature raises, different changes take place at the molecular level (nucleic acids and some proteins might be affected), which alter the cellular metabolism as well as may produce tissue damage in in vivo conditions [120]. Photons need to be administered in a well-optimized manner, where the exact degree of heating for a particular wavelength, duration, and power of light is experimentally determined. Light may also have non-thermal effects on cells, such as activation of light-sensitive molecular pathways, for example opsinmediated signaling, in particular when using animal models [121–124] or, one day, in human patients [125]. Most signaling networks have feedback loops to avoid over-excitability, and since these are often bypassed by optogenetic tools there is a risk to cause excitotoxicity – cell death caused by the over-activation and/or sustained activation of stimulatory signaling. This might be exacerbated in cases where photoactivating modules require long exposure times

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due to their reversibility. Although, this has been, at least partially, solved by the use of intermittent short flashes instead of long continuous exposure. Moreover, optogenetic tools are developing rapidly into more sensitive modules with better kinetics. At the end of the day, cellular off-target effects after photo-stimulation must be monitored by comparisons with untransfected control cells equally exposed [117].

6. Expert opinion Light is able to overcome some of the limitations that currently exist in drug discovery thanks to its remarkable precision. Moreover, light comes in a variety of colors (wavelengths), each with different properties such as penetration and interactions with cellular components and optogenetic modules. The discovery of large proteins, or protein domains, that respond to light is revolutionizing biomedical sciences.

Figure 5. Validation of drug targets by optogenetic actuators. (a) Identification of the molecular targets related to cell migration. Co-culture of the opto-modulator harboring cells (red) with control cells (blue) are treated by a small compound. The specificity of a drug target on the migratory phenotype can be either rescue or analyzed for an additive/synergetic effect due to the activation or inhibition of a particular signaling pathway. The readout is the quantification of migration of control vs photo-activated cells. (b) By photo-activating or inhibiting different signaling pathways, it is possible to study the specificity of drug targeting. For example, the expected effect of a drug targeting a receptor, would be a rescue by optogenetic activation of downstream signaling pathways. Moreover, the inhibition of one or several signaling pathways allows the careful dissection of which pathways should be targeted and which are side-effects. Same cells either having an optogenetic modulator (red cells) our without (blue) are co-cultured. A small compound is tested for its effects on cells e.g. induction of apoptosis (blebbing cells), and the specificity and rescue is measured. This set up can determine the involvement of other pathways in drug efficiency. C) Location of the molecular mechanism of drug resistance; Cells expressing opto-activators of a synthetic gate, with three possible factor outputs each i.e. a drug resistance gene (orange, yellow or green cells), are co-cultured together with population of control cells (blue) in the presence of a small compound. Only cells with relevant photoinduced drug resistance gene/s will survive, which can be easily quantified by the use of reporter genes. This approach enables the prediction of the mechanism of the drug resistance development. Inset) The creation of synthetic gene circuits which could be used to activate multiple gene expression outcomes from a couple of stimulations, in this case by different wavelengths of light. Inset is based in a chemical-induced double recombinase input (130), but can be used to activate or repress multiple combinations of genes responsible for specific biological processes e.g. drug resistance or metastasis.

EXPERT OPINION ON DRUG DISCOVERY

Optogenetic systems, originally devised for neurobiology, is expanding into many other fields, including oncology and drug discovery. In drug discovery, there are several optogenetic readout assays commonly used, e.g. reporter genes for the activation of growth or transcription factors, caspases, or the use of viability dyes. These assays, while useful in their own right, provide little information on the molecular mechanism(s) for drug action. Conversely, opto-actuators, which allow the rescue of the cell phenotype by activating/inhibiting a single molecular event (pathway or molecule), are inexpensive and tests could be run in the same platform, where control cells and cells carrying opto-devises, plus a fluorescent marker, are admixed in equal number and co-cultured, followed by e.g. migration (scratch) (Figure 5(a)) or viability (Figure 5(b)) assays once a small compound is added. The migration/ viability of cells will be quantified after light activation and small molecule incubation, where if the effect only occurs in the control cells, it can be attributed to either compensation by this pathway (resistance) or direct effect on the pathway – since many of the opto-moieties are originated from plant or bacterial proteins that are significantly different to those of humans. Another major problem that could be solved using novel optogenetical-based approaches, is the development of drug resistance by tumor cells, as cells usually use a ‘set’ of drug resistance genes [126]. Optogenetic systems can accomplish fast screenings through the activation of these genes – either one by one, or by several at once through i.e. photoactivatable-CRISPR/dCas9 (Figure 5(c)). Thereby, such rapid screening for potential drug resistance pathways will be highly beneficial, as new drugs could then be coadministrated with drugs acting in such pathways, or be a method for the discovery of such drugs. Moreover, any small changes in culturing conditions and toxicity will be obliterated as control and treated cells will be in the very same conditions (Figure 5). Similarly, the molecular activity e.g. cell death, can be determined by rescuing experiments of single molecular pathways by use of co-cultures of the same cell type, one of which carries a photoactivatable effector. A variety of such effectors which allow manipulation of cellular processes or (epi)genetic changes exist, covering many of the cancer discovery pipeline targets. Optogenetic actuators in combination with synthetic gene circuits allow multiple outputs from a limited number of inputs. To exemplify this, imagine two opto-recombinases, each is activated by a different wavelength; dependent on whether one, or both, are activated and the structure of the synthetic gate (4 sequential genes separated by the recognition sequences of the recombinases), there are 4 potential outputs [127]. As such, in just one experiment 4 targets could be tested with nothing other than light (inset in Figure 5). Likewise, we can rationalize that light responsive systems affecting different signaling cascades can be controlled separately or simultaneously to determine the effect of activation of compensatory pathways during drug testing. However, challenges remain to be solved in the optogenetic field and in any case, most, if not all, light-inducible

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systems leak in darkness, and some are quickly reversible which could be beneficial in some cases, although might require long exposition times – risking phototoxicity, and making them unpractical to in vivo settings. Many of the photo-responsive moieties are large proteins that not only challenge the potential problem of gene delivering to cells, but they surely have unforeseen interactions with cellular components. Nevertheless, optogenetics is already expanding our understanding of biological processes in health and disease conditions. These tools can be used synergistically, orthogonally or antagonistically to one another and to other optical systems, fluorescent proteins or dyes, and in conjunction with synthetic biology circuitry. Although to date, no such combinations have been attempted, we expect some very exciting years ahead in opto-supported drug discovery, cancer biology and thus, the overall of cancer therapy. The transition from in vitro to in vivo, e.g. animal models, of optogenetic tools is rather straightforward as can be seen in the multiple examples mentioned in this work. Moreover, the delivery of light in vivo is minimally invasive such as by implanted micro-LED lights which can be wirelessly activated [128], or by tampered optical fibers allowing spatiotemporal tissue illumination [129]. The strategies of light delivery to the right body location is a critical issue, and thus under continuous development, a widely discussed issue in both research and clinical contexts [130–132]. In summary, we envision that the optogenetical tools reviewed here, and those to be developed in near future, will become an integral part of drug discovery as well as cancer research, as they are able to overcome some of the crucial disadvantages of the methodologies currently in use.

Funding The authors are supported by grants: 1) DEC-2015/17/B/NZ1/01777; 2) DEC-2017/01/X/NZ1/00107 and 3) DEC-2017/25/B/NZ4/02364 from the Polish National Science Centre (NCN).

Declaration of Interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Peer reviewers on this manuscript have no relevant financial or other relationships to disclose

ORCID Michał Kiełbus http://orcid.org/0000-0001-6156-6217 Jakub Czapiński http://orcid.org/0000-0001-6928-6359 Adrian Odrzywolski http://orcid.org/0000-0002-3370-8801 Grażyna Stasiak http://orcid.org/0000-0002-8488-5381 Kamila Szymańska http://orcid.org/0000-0001-8892-1290 Joanna Kałafut http://orcid.org/0000-0002-7452-8874 Michał Kos http://orcid.org/0000-0001-5557-7892 Krzysztof Giannopoulos http://orcid.org/0000-0003-0135-4030 Andrzej Stepulak http://orcid.org/0000-0002-1872-394X Adolfo Rivero-Müller http://orcid.org/0000-0002-9794-802X

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