cells Review Functional Genomics Approaches to Elucidate Vulnerabilities of Intrinsic and Acquired Chemotherapy Resistance Ronay Cetin 1,† , Eva Quandt 2,† and Manuel Kaulich 1,3,4,* 1 Institute of Biochemistry II, Goethe University Frankfurt-Medical Faculty, University Hospital, 60590 Frankfurt am Main, Germany; [email protected] 2 Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, 08195 Barcelona, Spain; [email protected] 3 Frankfurt Cancer Institute, 60596 Frankfurt am Main, Germany 4 Cardio-Pulmonary Institute, 60590 Frankfurt am Main, Germany * Correspondence: [email protected]; Tel.: +49-(0)-69-6301-5450 † These authors contributed equally to this work. Abstract: Drug resistance is a commonly unavoidable consequence of cancer treatment that results in therapy failure and disease relapse. Intrinsic (pre-existing) or acquired resistance mechanisms can be drug-specific or be applicable to multiple drugs, resulting in multidrug resistance. The presence of drug resistance is, however, tightly coupled to changes in cellular homeostasis, which can lead to resistance-coupled vulnerabilities. Unbiased gene perturbations through RNAi and CRISPR technologies are invaluable tools to establish genotype-to-phenotype relationships at the genome scale. Moreover, their application to cancer cell lines can uncover new vulnerabilities that are associated with resistance mechanisms. Here, we discuss targeted and unbiased RNAi and CRISPR efforts in the discovery of drug resistance mechanisms by focusing on first-in-line chemotherapy and their enforced vulnerabilities, and we present a view forward on which measures should be taken to accelerate their clinical translation. Citation: Cetin, R.; Quandt, E.; Kaulich, M. Functional Genomics Keywords: chemotherapy resistance; cancer and drug vulnerabilities; functional genomics; RNAi Approaches to Elucidate Vulnerabilities of Intrinsic and and CRISPR screens Acquired Chemotherapy Resistance. Cells 2021, 10, 260. https://doi.org/ 10.3390/cells10020260 1. Introduction Academic Editor: Martin Michaelis The administration of single anti-cancer drugs sooner or later selects for the occurrence Received: 15 January 2021 and outgrowth of drug-resistant cancer cell populations, with therapy failure and disease Accepted: 25 January 2021 relapse being the ultimate consequences. Focusing on chemotherapeutics, this led to Published: 28 January 2021 the development of multidrug treatment protocols in which agents with different modes of actions are combined with the aim to suppress the occurrence of drug resistance. In Publisher’s Note: MDPI stays neutral hematological disorders, such as Hodgkin’s lymphoma and acute lymphoblastic leukemia, with regard to jurisdictional claims in multidrug regimens, such as ABVD (doxorubicin-Adriamycin, bleomycin, vinblastine, published maps and institutional affil- and dacarbazine) or CHOP (cyclophosphamide, hydroxydaunorubicin, vincristine sulfate- iations. oncovin and prednisone), when provided to patients with early-stage tumors, can result in 5-year progression-free survival of 80–98%, with many patients being cured. However, for most, if not all other solid and non-solid malignancies, therapy success with multidrug regimens remains to be the exception. Copyright: © 2021 by the authors. Resistance can be restricted to a specific drug, or affect different drugs with indepen- Licensee MDPI, Basel, Switzerland. dent modes of action, named multidrug resistance (MDR). However, even in non-solid This article is an open access article tumors, chemotherapeutic multidrug regimes result in the appearance of drug-resistant cell distributed under the terms and populations, containing pre-existing (intrinsic) and newly acquired resistance mechanisms conditions of the Creative Commons that can be mechanistically separated, as summarized in Figure1. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cells 2021, 10, 260. https://doi.org/10.3390/cells10020260 https://www.mdpi.com/journal/cells Cells 2021, 10, 260 2 of 27 Cells 2020, 9, x FOR PEER REVIEW 2 of 26 Figure 1. Mechanisms contributing to chemoresistance include molecule transporters that increase the drug efflux, reduc- Figure 1. Mechanisms contributing to chemoresistance include molecule transporters that increase the drug efflux, reducing ing their intracellular concentrations; higher proliferation induced by oncogene activation or mutations in tumor suppres- their intracellular concentrations; higher proliferation induced by oncogene activation or mutations in tumor suppressor sor genes; deregulation of apoptosis and metabolic reprogramming due to mitochondrial alteration; invasive phenotypes causedgenes; deregulationby overexpression of apoptosis of stem andcell markers; metabolic existence reprogramming of inherently due toresistant mitochondrial cell subpopulations alteration;invasive which present phenotypes a cer- taincaused degree by overexpressionof quiescence and of stema high cell expr markers;ession of existence stem cell of markers inherently, as well resistant as drug cell efflux subpopulations and anti-apoptotic which presentproteins; a elevatedcertain degree secretion of quiescence of exosomes and by a tumor high expression cells, which of mediate stem cell the markers, transfer as of well cargos as drug that effluxcan promote and anti-apoptotic resistance by proteins; several mechanismselevated secretion (e.g., ofgrowth exosomes advantage, by tumor drug cells, efflux); which pro mediate-survival the function transfer, ofmediated cargos that by increased can promote autophagic resistance activity by several; the activationmechanisms of alternative (e.g., growth DNA advantage, repair pathways. drug efflux); pro-survival function, mediated by increased autophagic activity; the activation of alternative DNA repair pathways. Intrinsic resistance may be defined as the pre-existence of resistance mechanisms be- fore therapyIntrinsic is resistance initiated. mayThe reasons be defined are asheterogeneous the pre-existence and ofinclude resistance (1) the mechanisms pre-existence be- offore therapy therapy-resistant is initiated. cell populations; The reasons are (2) heterogeneouslow therapy tolerance and include of the (1) patient the pre-existence or the occur- of rencetherapy-resistant of unbearable cell side populations;-effects; (3) (2) an low inability therapy of tolerance the therapy of the to patient reach orits theneeded occurrence phar- macokineticof unbearable profile side-effects; by means (3) of an altered inability absorption, of the therapy distribution, to reach metabolism, its needed pharmacoki-or excretion. Innetic contrast profile to byintrinsic means mechanisms, of altered absorption, acquired resistance distribution, may metabolism,be defined by or the excretion. appearance In ofcontrast drug-resistant to intrinsic cell mechanisms, populations acquired containing resistance secondary may begenetic defined modifications by the appearance acquired of duringdrug-resistant the course cell populationsof therapy, ultimately, containing secondaryas with intrinsic genetic resistance, modifications leading acquired to therapy during failure.the course Acquired of therapy, resistance ultimately, mechanisms as with include, intrinsic but resistance, are not limited leading to (1) to therapyincreased failure. rates ofAcquired drug efflux resistance or decreased mechanisms rates include,of drug butinflux are into not limitedthe tumor to (1) cells, increased mediated rates by of trans- drug membraneefflux or decreased transporters rates of of drug drug uptake influx and/or into the efflux; tumor (2) cells, biotransformation mediated by transmembrane and drug me- tabolism,transporters mainly of drug due uptaketo CYPs and/or (Cytochromes efflux; (2) P450s) biotransformation in the tumor; (3) and altered drug role metabolism, of DNA repair and impairment of apoptosis; (4) role of epigenetics/epistasis by methylation, acet- Cells 2021, 10, 260 3 of 27 mainly due to CYPs (Cytochromes P450s) in the tumor; (3) altered role of DNA repair and impairment of apoptosis; (4) role of epigenetics/epistasis by methylation, acetylation, and altered levels of microRNAs leading to alterations in upstream or downstream ef- fectors; (5) mutation of drug targets in targeted therapy and alterations in the cell cycle and its checkpoints; (6) the tumor microenvironment. Importantly, cancers can become chemotherapy resistant by combinations of these mechanisms. For instance, the action of methotrexate depends on its active transport into cells through the reduced-folate trans- porter 1 (RFT-1), its conversion to a long-lived intra-cellular polyglutamate, and its binding to the dihydrofolate reductase (DHFR), which leads to the inhibition of the synthesis of thymidylate and purines and the induction of apoptosis. Cellular defects in any of these steps can lead to drug resistance. Mutations in RFT-1, amplification or mutation of DHFR, loss of polyglutamation, and defects in the apoptotic pathway have all been shown to lead to the loss of efficacy of methotrexate [1,2]. Anti-cancer drug resistance mechanisms, however, can be accompanied by the emer- gence of new and therapy-restricted vulnerabilities. For example, resistance can arise as a compensation for the effects of treatment due to the “addiction” of cancer cells to a specific oncogene. Functional genetic screens have been used to identify such acquired vulnerabili- ties in several cancer cell lines [3,4]. These dependencies,