The inhibition of transcription and translation to prevent neoplastic cells proliferation

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Methods to assess immunogenic cell death

In anticancer therapy, all aforementioned ICD hallmarks, including the eIF2 -dependent exposure of CALR, autophagy-mediated ATP release, the secretion of HMGB1 into the extracellular space, type I IFN responses as well as ANXA1 release, are essential for ICD induction and adaptive immune response mounting. However, the mere presence of the molecules cannot predict with an absolute certainty the immunogenicity of cell death, underlining the fact that other immunogenic pathways remain to be discovered (Galluzzi et al, 2017).
The current gold standard approach to evaluate immunogenic cell death relies on in vivo vaccination assay employing immunocompetent animals. The experiment consists in injection of murine cells treated in vitro with a cytotoxic agent into syngeneic mice, followed by rechallenge with living cells of the same type (one to two weeks later). The absence of tumors or delayed tumor growth indicates that an adaptive immune response has been elicited by the cells dying in response to the employed cytotoxicant (Humeau et al, 2019; Kepp et al, 2014). Of note, the standard ICD inducers MTX and doxorubicin vaccinate around 80 % mice from CT26 colon carcinoma or MCA205 fibrosarcoma (Humeau et al, 2019; Kepp et al, 2014). In case of agents or combinations of drugs that enable the cure from transplanted cancers, the absence of proliferation of further injected cells of the same type also proves that immune memory has been elicited. The immunogenicity of such agents can be further confirmed by comparing their effect in immunocompetent versus immunodeficient mice. However, compounds that are endowed with immunostimulatory effect also exhibit decreased effectivity in immunodeficient mice, yet do not necessarily induce pre-mortem signals leading to the release of DAMPs from cancer cells, emphasizing the limitations of this technique to determine if an agent induces ICD. These methods are constrained by the restricted amount of available syngeneic models, as well as cost, time and ethical issues. As an alternative to vaccination, in vitro phagocytosis assay can be performed to assess the recruitment of DCs by dying tumor cells. Of note, this method does not test for the further activation of adaptive immune responses. Unfortunately, the aforementioned current available techniques do not enable to study ICD in human cells.
Regarding the growing importance of ICD for anticancer therapies, there is a need to discover new agents endowed with the capacity to elicit such process. We therefore investigated if we could predict in silico the immunogenic potential of drugs, based on their chemical and physical properties, such as molecular weight, number of hydrogen bonds donors/acceptors, polar surface area etc. Using machine learning approaches and taking into account that anthracyclines are strong ICD inducers, a model calculating an “ICD score” according to molecular properties was established (Figure 7) (Bezu et al, 2018a). This model was further applied to two independent chemical compound libraries, namely the US Drug Set of Food and Drug Administration (FDA)-approved drugs and the National Cancer Institute (NCI) mechanistic diversity set, returning the highest ICD scores for drugs previously shown as exhibiting hallmarks of ICD in vitro (Bezu et al, 2018a; Menger et al, 2012; Sukkurwala et al, 2014a). Hence, this algorithm is endowed with high accuracy and offers the opportunity to quickly predict new potential ICD inducers among huge libraries.

Immunogenic cell death inducers

The first anticancer agents discovered as intrinsically endowed with the ability to elicit a proinflammatory immune response in mice were doxorubicin, daunorubicin and iadarubicin. Indeed, when dying cancer cells pre-treated with these agents are injected in mice, they induce long term protection against subsequent challenge with living cells of the same type, whereas cancer cells displaying the same extent of apoptosis following mitomycin C treatment or freeze thawing cycles are not able to do so (Casares et al, 2005). Yet another anthracycline MTX (Obeid et al, 2007a, Panaretakis et al, 2009), as well as the platinum derivative OXA (Ma et al, 2011; Obeid et al, 2007a; Panaretakis et al, 2009; Schiavoni et al, 2011), the alkylating agent cyclophosphamide (Chen et al, 2012; Ghiringhelli et al, 2009; Schiavoni et al, 2011), the proteasome inhibitor bortezomib (Chang et al, 2012; Cirone et al, 2012; Demaria et al, 2005; Spisek et al, 2007), the RNA polymerase II inhibitor lurbinectedin (Xie et al, 2019a), the cyclin dependent kinase inhibitor dinaciclib (Hossain et al, 2018), the topoisomerase inhibitor teniposide (Wang et al, 2019b), the bromodomain inhibitor JQ1 (Riganti et al, 2018; Wang et al, 2019a) and the antibiotics bleomycin (Bugaut et al, 2013), wogonin (Yang et al, 2012) and septacidin (Sukkurwala et al, 2014a) share the same properties. Some anticancer therapies involving physical signals such as radiotherapy (Apetoh et al, 2007; Ma et al, 2011; Obeid et al, 2007a), photodynamic therapy (Garg et al, 2012a; Gomes-da-Silva et al, 2018; Korbelik & Dougherty, 1999; Korbelik et al, 2007; Korbelik et al, 2011; Krosl et al, 1995), ultraviolet C (UVC) light (Obeid et al, 2007a; Panaretakis et al, 2009; Schiavoni et al, 2011; Yamamura et al, 2015), electrical pulses (Nuccitelli et al, 2015; Nuccitelli et al, 2017), high hydrostatic pressure (Fucikova et al, 2014; Urbanova et al, 2017), microwave thermal ablation (Yu et al, 2014) and photochemotherapy (Tatsuno et al, 2019; Ventura et al, 2018) have also shown to be immunogenic, as well as some targeted agents like targeting epidermal growth factor receptor (EGFR) antibody (Pozzi et al, 2016); (Garrido et al, 2011), certain oncolytic peptides (Zhou et al, 2016a), oncolytic viruses (Bommareddy et al, 2019; Koks et al, 2015; Zamarin et al, 2014) and certain specific bacterial toxins (Sun et al, 2015) (Table 1).
In most cases, chemotherapies that are non-immunogenic fail to induce CALR exposure, such as etoposide, mitomycin C or cisplatin (CDDP) (Martins et al, 2011; Obeid et al, 2007b). Their immunogenic potential can be restored by combination with agents targeting the endoplasmic reticulum (ER) and consequently activating CALR translocation, like the ER stress inducer thapsigargin or the eIF2 phosphatase inhibitor salubrinal (Obeid et al, 2007b). Crizotinib (Liu et al, 2019b) and cardiac glycosides (Menger et al, 2012), even though eliciting all the hallmarks of ICD in vitro, were efficient ICD inducers only when combined with cytotoxic agents like CDDP or mitomycin C.
Many other agents trigger the ICD-pathognomonic eIF2 phosphorylation (Bezu et al, 2018a), elicit the exposure and release of certain DAMPs (CALR, ATP, HMGB1, ANXA1), activate some immune cells (DCs, NK, NKT, CD8+ T cells), inhibit immunosuppressive cells (Tregs, MDSCs) and/or promote proinflammatory cytokines secretion (typically type I IFNs, IL1- , IFN or IL-6) (Table 1). Even if this suggests an immunogenic potential, it is not sufficient to ensure that they bona fide induce ICD. Agents that induce a partial immune response may synergize to efficiently trigger an anticancer immune response, like (i) 5-Fluorouracil and cycloheximide, which together are very efficient in reducing the proliferation of the thymoma cell line EL-4, (effect abolished in nu/nu mice) (Vincent et al, 2010), (ii) gemcitabine and the hypoxia inducible factor-1 (HIF-1) inhibitor, which can induce vaccination in pancreatic ductal adenocarcinoma (Zhao et al, 2015), (iii) PX-478 or temozolomide with oncolytic adenovirus, which together could induce long term protection against prostate cancer in mice, accompanied by an activation of CALR exposure as well as ATP and HMGB1 release (effect even amplified by addition of cycloheximide) (Liikanen et al, 2013). In addition, different studies have shown that the synergistic anticancer effect of radiation therapy and chemotherapy relies on an activation of the immune system (Golden et al, 2014; Rubner et al, 2014).

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The anticancer agents dactinomycin inhibits transcription

Dactinomycin (DACT, also known as actinomycin D and called actinomycin C1 in the past) is a 1.26 kDa peptide belonging to the family of actinomycins (Figure 8), which are characterized by their two pentapeptide lactones linked to a phenoxazinone dicarbolxylic acid and appear as clear yellow liquids. They are produced by different strains of Streptomyces bacteria and were first isolated by Selman Waksman and H. Boyd Woodruff in 1940 (Waksman & Woodruff, 1940), with more than 40 actinomycin reported today. The group of Waksman has demonstrated the antibacterial and anticancer effects of DACT, further confirmed by several studies on sarcoma cell lines (Gregory et al, 1956; Reilly et al, 1953). This led to the approval of DACT by the FDA in 1964, constituting the first antibiotic used as an anticancer agent. DACT is commercialized under the name Cosmegen and is mostly obtained by fermentation of Streptomyces parvullus, even if it was recently shown that Streptomyces flavogriseus also enables to obtain high yield of DACT (Wei et al, 2017). It is currently part of chemotherapeutics combinations for the treatment of pediatric sarcoma: Wilms’ tumors, rhabdomyosarcoma, and Ewing’s sarcoma, for trophoblastic neoplasia, as well as for some cases of advanced testicular cancers, and is still subject of numerous in vitro, in vivo and clinical studies. Its anticancer effect is thought to be due to intercalation between the DNA helix leading to transcription inhibition, even though other effects on cells have been enlightened. It is also widely used in laboratories for its ability to inhibit transcription.

Transcription and translation in eukaryotic cells

Transcription is the synthesis of single stranded RNA from double stranded DNA (dsDNA) in the nucleus of cells, using the template (non-coding) strand. It generates a 5’ to 3’ pre-RNA molecule, similar to the coding strand apart from thymines which are replaced by uracils. Transcription is catalyzed by RNA polymerases (RNAP). There are three RNAP in the nucleus: RNAPI for 5.8S, 18S and 28S ribosomal RNA (rRNA) synthesis, RNAPII for mRNA and RNAPIII for transfer RNA (tRNA), 5S rRNA and other short RNAs transcription (Table 2), in addition to a mitochondrial-specific polymerase. All three nucleic polymerases contain ten common subunits forming the catalytic subunit core, two additional more distant subunits, as well as peripheral subunits for RNAPI and III. While RNAPII and III are located into the nucleoplasm, RNAPI operates within the nucleoli, which are located around tandem repeats of rDNA called nucleolus organizer regions (NOR). rRNA transcription occurs in fibrillar centers, then pre-rRNA is processed in dense fibrillar region involving the proteins fibrillarin and nucleolin and finally the 40S and 60S subunits of the pre-ribosome are assembled into granular components by nucleophosmin (Figure 9) (Pombo et al, 1999).

Table of contents :

ACKNOWLEDGEMENTS
PREAMBLE
CONTENT
ABBREVIATIONS
INTRODUCTION
1. Cancer
1.1. Overview
1.2. Mechanisms of oncogenesis
2. Role of the immune system in antitumor therapy
2.1. From the discovery of tumor antigens to the development of immunotherapies
2.2. Mechanisms of antitumor immunity
2.3. Immunoediting accompanies cancer progression
2.4. Adjuvanticity through immunogenic cell death
2.4.1. Mechanisms of chemotherapeutics-driven immunogenic cell death .
2.4.1.1. eIF2 phosphorylation-dependent calreticulin exposure
2.4.1.2. Autophagy-mediated ATP secretion
2.4.1.3. HMGB1 release and TLR4 mimicry
2.4.1.4. Autocrine signaling of type I interferon
2.4.1.5. The ANXA1 FPR1 axis
2.4.2. Methods to assess immunogenic cell death
2.4.1. Immunogenic cell death inducers
3. The anticancer agents dactinomycin inhibits transcription
3.1. Transcription and translation in eukaryotic cells
3.1.1. Mechanisms of transcription
3.1.2. Mechanisms of translation
3.1.2.1. From mRNA to protein
3.1.2.2. ER stress inhibits cap-dependent translation
3.1.3. The inhibition of transcription and translation to prevent neoplastic cells proliferation
3.2. Dactinomycin intercalates into the DNA and inhibits transcription
3.3. Other anticancer mechanisms of dactinomycin
3.3.1. Dactinomycin is a topoisomerase inhibitor
3.3.2. Dactinomycin inhibits protein synthesis
3.3.3. Dactinomycin induces apoptosis
3.3.4. Dactinomycin induces photosensitization
3.3.5. Dactinomycin inhibits respiration and glycolysis
3.3.6. The effect of dactinomycin on the immune system
3.4. Pharmacokinetics of dactinomycin
3.5. Dactinomycin in the clinic
3.5.1. Cancers treated with dactinomycin-based chemotherapy
3.5.2. Clinical trials involving dactinomycin
AIM OF THE WORK
MATERIAL AND METHODS
RESULTS
Identification of dactinomycin as a bona fide ICD inducer.
Immune-dependent anticancer effects of dactinomycin.
Inhibition of transcription by a panel of ICD inducers.
Inhibition of transcription as an ICD hallmark.
SUPPLEMENTARY FIGURES AND TABLES
DISCUSSION
PERSPECTIVES
COLLABORATIONS
BIBLIOGRAPHY

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