Treating hematological malignancies with drugs inhibiting ribosome biogenesis: when and why

Treating hematological malignancies with drugs inhibiting ribosome biogenesis: when and why It is well known that chemotherapy can cure only some cancers in advanced stage, mostly those with an intact p53 pathway. Hematological cancers such as lymphoma and certain forms of leukemia are paradigmatic examples of such scenario. Recent evidence indicates that the efficacy of many of the alkylating and intercalating agents, antimetabolites, topoisomerase, and kinase inhibitors used in cancer therapy is largely due to p53 stabilization and activation consequent to the inhibition of ribosome biogenesis. In this context, innovative drugs specifically hindering ribosome biogenesis showed preclinical activity and are currently in early clinical development in hematological malignancies. The mechanism of p53 stabilization after ribosome biogenesis inhibition is a multistep process, depending on specific factors that can be altered in tumor cells, which can affect the antitumor efficacy of ribosome biogenesis inhibitors (RiBi). In the present review, the basic mechanisms underlying the anticancer activity of RiBi are discussed based on the evidence deriving from available preclinical and clinical studies, with the purpose of defining when and why the treatment with drugs inhibiting ribosomal biogenesis could be highly effective in hematological malignancies. Keywords: Ribosome biogenesis inhibitors, Chemotherapy, Lymphoma, Leukemia, Ribosomal proteins, MDM2, p53, pRb Background selectively hindering the transcription of ribosomal (r) The ribosome biogenesis is defined as the process of RNA, thus inhibiting ribosome biogenesis without hav- building new ribosomes, the intracellular organelles ing genotoxic effects, have been proposed as a new where protein synthesis takes place. therapeutic approach, based on p53 activation [7–12]. In recent years, several studies on the relationship be- However, it is known since long time that chemotherapy tween cell growth and proliferation produced important can cure only some cancers once they reach advanced data regarding the mechanisms linking ribosome biogen- stages. In fact, despite initial responses, the majority of esis, which is at the basis of cell growth, to the progression metastatic solid tumors ultimately progress under chemo- through the cell cycle phases of the proliferating cell. There therapy treatment. Hematological malignancies (such as is now evidence that a perturbed ribosome biogenesis acti- lymphomas and acute leukemias) represent paradigmatic vates a pathway leading to the stabilization and activation examples of the few cancers that can be cured by chemo- of the tumor suppressor protein p53, which in turn induces therapeutic agents and will be the main topic of the cell cycle arrest and/or apoptotic cell death [1–4]. present review [13]. The basic biological characteristic Current evidence indicates that inhibition of ribosome underlying the intrinsic curability of such cancers is that, biogenesis represents a major mechanism by which in a significant fraction of cases, they retain a functional many of the currently used chemotherapeutic drugs p53-mediated response to nucleolar stress arising from (alkylating and intercalating agents, antimetabolites, ribosomal biogenesis inhibition; on the other hand, as a topoisomerase inhibitors) exert their cytotoxic activity matter of fact, the presence of genomic alterations of the on cancer cells [5, 6]. Importantly, a series of new drugs TP53 gene is an established negative prognostic predictor in lymphoma, acute and chronic leukemias treated with chemotherapy regimens [14–17]. * Correspondence: enrico.derenzini@ieo.it; davide.trere@unibo.it European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy DIMES, Università di Bologna, Via Massarenti 9, Bologna, Italy © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 2 of 13 Since p53 stabilization and activation is a multistep binding factor (UBF), are present [33]. For the transcrip- and tightly regulated process, in principle, the prerequis- tion of the 5S rRNA by Pol III, the transcription factors ite for the antitumor efficacy of drugs inhibiting ribo- TFIIIC and TFIIIB are necessary [34–36]. In proliferat- some biogenesis should be the presence in the tumor ing cells, the rate of ribosome biogenesis is enhanced in cells, other than a normally functioning p53, also of order to assure an adequate ribosome complement for those factors necessary for the activation of p53 and the the daughter cells and inhibition of ribosome biogenesis induction of a p53-mediated cell cycle arrest and/or the arrests cell cycle progression [37]. Furthermore, the rate apoptosis. These factors, which control cell cycle pro- of ribosome biogenesis influences the length of the cell gression in normally proliferating cells [18], are qualita- cycle: higher the level of ribosome biogenesis, more tively and quantitatively altered in the large number of rapid the cell cycle progression [38]. Ribosome biogen- cancers [19, 20], thus influencing the sensitivity to ribo- esis rate in cancer shows high variability, depending on a some biogenesis (RiBi) inhibitors. multiplicity of factors including the activation of specific Therefore, it seems timely to critically review the char- intracellular signaling pathways and deregulated activity acteristics of cancer cells which affect their sensitivity to of oncogenes and tumor suppressors. On the other RiBi inhibitors, with the purpose of highlighting those hand, quantitative and qualitative changes in ribosome parameters which render the treatment with these drugs biogenesis have been shown to facilitate neoplastic appropriate or not in hematological malignancies. For transformation. For a detailed description of the the convenience of the reader, the normal process of relationship between ribosome biogenesis and cancer, ribosome biogenesis will be first briefly described. the reader should refer to [39–44]. In hematological malignancies, such as aggressive lymphoproliferative Ribosome biogenesis neoplasms, it is worth mentioning the oncogenic Ribosomes are ribonucleoprotein particles which are lo- cooperation between the MYC oncogene and the cated in the cytoplasm where, either free or membrane- phosphatidyl-inositol-3-kinase (PI3K) signaling pathway bound, are engaged in protein synthesis. Four types of [45], which converge in stimulating rRNA synthesis and ribosomal RNA (rRNA) molecules and about 80 differ- ribosome biogenesis [46]. ent ribosomal proteins constitute the ribosome. Ribo- some formation occurs mainly in the nucleolus, being Inhibition of ribosome biogenesis activates the later completed in the nucleoplasm and in the cytoplasm RPs/MDM2/p53 pathway (see for reviews: [21–24]). In the nucleolus, ribosomal Available data indicate that the levels of p53 expression genes are transcribed by RNA polymerase I (Pol I) to and activity are mainly regulated by interactions with generate the 47S rRNA precursor, which undergoes to the tumor suppressor MDM2 (murine double minute 2, site-specific methylation and pseudo uridylation, and and HDM2 in humans). MDM2 is an E3 ubiquitin ligase processing to give rise to the mature 18S, 5.8S, and 28S which negatively controls p53 activity in two ways: by rRNA. The fourth types of rRNA, the 5S rRNA, is syn- binding to the protein and inhibiting its transactivation thesized in the nucleoplasm by RNA polymerase III (Pol activity, and by facilitating its proteasome degradation III) and then imported in the nucleolus together with [47–49]. In normal proliferating cells, the level of p53 is the ribosomal proteins (RPs), whose mRNA is tran- maintained low because of the binding with MDM2 with scribed by RNA polymerase II (Pol II). The assembling of consequent p53 ubiquitination and proteasome digestion rRNA molecules with the RPs constitutes the two sub- [50]. When a perturbation in the ribosome biogenesis units of the mature ribosome, the large 60S and the small occurs (ribosome stress), it results in the binding of sev- 40S subunit. The large 60S subunit is constituted by one eral ribosomal proteins, no longer used for ribosome each of the 28S, 5.8S, and 5S RNA molecules, together building, to MDM2. This binding relieves the inhibitory with 47 ribosomal proteins (RPLs); the small 40S subunit activity of MDM2 toward p53 (see reviews [2–4, 51, 52]) contains only one 18S RNA molecule and 33 ribosomal (Fig. 1). Although there is evidence that RPL5, RPL11, proteins (RPSs) [25, 26]. Both subunits migrate from the and RPL23 play a major role in neutralization of MDM2 nucleolus to the cytoplasm where they form the 80S ribo- activity and in the induction of p53 stabilization [50, 53– some particle. In the process of ribosome biogenesis, more 58], the list of ribosomal proteins (of both large and than 150 non-ribosomal proteins and around 70 small nu- small ribosomal subunit) able to inhibit MDM2 activity cleolar RNAs are involved [27–32]. and to stabilize p53 upon “ribosomal stress” is rapidly For the transcription of the of 47S pre-rRNA, the as- expanding [52]. For a valid binding to MDM2 and its in- sembly of a specific multiprotein complex at the rDNA activation, the RPL11 and RPL5 must form a complex promoter containing Pol I is required. In this complex, with the 5S rRNA and all the components of this com- three basal factors, termed transcription initiation factor plex are necessary for its inhibitory function [59, 60]. I (TIF-I) A, selectivity factor 1 (SL1), and upstream p53 stabilization always causes cell cycle arrest in Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 3 of 13 MDM2 phosphorylation results in increased stability of MDM2 with consequent p53 degradation [69, 70]. As mentioned before, constitutive PI3K signaling is common in lymphoproliferative neoplasms, and PI3K inhibitors are in clinical development in lymphoid cancers. These no- tions could be relevant for designing therapeutic combin- ation strategies aimed at increasing the p53-mediated response to the inhibition of ribosome biogenesis. Development of selective inhibitors of ribosome biogenesis As briefly mentioned before, a strong contribution to p53 activation induced by chemotherapeutic agents is due to the inhibition of ribosomal biogenesis. As re- ported by Burger et al. [5], a series of drugs currently used for treating solid cancers and hematological malig- nancies inhibit ribosome biogenesis at the level of rRNA transcription and/or at the level of rRNA processing (Table 1). To this list, cyclophosphamide and mycophe- Fig. 1 Schematic representation of the pathway activated by drug- nolic acid should be added. Cyclophosphamide, a widely induced perturbation of rRNA synthesis. Ribosomal proteins (RPs), no used anticancer drug, also inhibits rRNA transcription longer used for ribosome building, bind to MDM2, thus inhibiting its [71], after being converted to acrolein [72, 73], and the ubiquitin ligase activity toward p53 and the proteasome digestion of immunosuppressant mycophenolic acid has been dem- the tumor suppressor. As a consequence, p53 accumulates and induces onstrated to inhibit the synthesis of rRNA [74]. transcription of p21, PUMA, and BAX. P21 is responsible for the cell cycle arrest by hindering pRb phosphorylation: in fact, hypo-phosphorylated In recent years, several efforts have been made to de- pRb binds to and inhibits the activity of the transcription factor E2F1, velop specific inhibitors of ribosomal biogenesis, in order whose target gene products are necessary for cell cycle progression. to achieve a selective inhibition of rRNA synthesis without The induction of the pro-apoptotic factors PUMA and BAX activates the the genotoxic effects proper of chemotherapeutic drugs. process of apoptotic cell death In this light, it appears to be of particular relevance the CX-5461 molecule which selectively inhibits ribosome proliferating cells and, depending on the quantitative biogenesis, most likely by disrupting the SL-1/rDNA com- level of stabilized p53, also apoptotic cell death [61–63]. plex, promoting a cancer-specific activation of p53. Recent p53 arrests cell cycle progression by inhibiting the phos- preclinical data indicate high activity of CX-5461 in MYC- phorylation of the tumor suppressor retinoblastoma driven lymphoma, providing the rationale for further protein, pRb. In its hypo-phosphorylated form, pRb clinical development of this compound [7, 75, 76]. binds to and inhibits the activity of E2F1, a transcription CX-5361 is currently under phase I clinical trial for the factor whose target genes are necessary for cell cycle treatment of patients with advanced hematologic malig- progression. The inhibition of E2F1 activity by hypo- nancies, including acute myeloid leukemia. phosphorylated pRb reduces the expression of both Finally, there is experimental evidence that a small mo- cyclin E and A, necessary factors for cell cycle progres- lecular compound, BMH-21, and a small-molecule pep- sion from G1 to S phase and from G2 to M phase tide (22mer) also selectively inhibit rDNA transcription. respectively, with consequent cell accumulation in G1 BMH21 binds to GC-rich sequences and inhibits RNA and G2 phase [64]. The induction of apoptotic cell death Pol I activity [9]. It also induces the proteasome- by p53 is a consequence of induced expression of the dependent destruction of the large catalytic subunit in the pro-apoptotic members of the B cell lymphoma 2 (Bcl-2) Pol I complex, as do three other small molecular com- gene family, PUMA, and BAX [63, 65–67](Fig. 1). Finally, pounds, BMH-9, BMH-22, and BMH-23 [10]. The 22mer it should be noted that additional factors may interact targets the interface between RNA polymerase I and Rrn3, with the RPs/MDM2/p53 axis, such as the ARF tumor thus selectively inhibiting the synthesis of rRNA [11]. suppressor and the activation of the PI3K pathway. In fact, ARF loss is a common genetic event in cancer and espe- Factors determining cancer cell sensitivity to drugs cially in aggressive lymphoid neoplasms, resulting in in- inhibiting ribosome biogenesis creased MDM2 activity and increased p53 degradation The p53 status (reviewed in [68]). On the other hand, MDM2 is a down- Since a major effect of ribosome biogenesis inhibition is stream target of the PI3K-AKT axis, and AKT-induced the activation of p53, the cytostatic and cytotoxic effects Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 4 of 13 Table 1 Drugs used to treat hematological and solid with inactivated p53 in which the synthesis of rRNA was malignancies which are effective or highly effective in the hindered by polymerase I silencing [79]. Also, p53 silen- inhibition of rRNA transcription or processing (modified from cing significantly reduced the antiproliferative effects of Burger et al., 2010) [5] 5-fluorouracil and methotrexate or doxorubicin, in human inibition of rRNA synthesis cancer cell lines harboring wild type (wt) p53 [78]and transcription processing treatment of human leukemia and lymphoma cell lines Alkylating agents: with CX-5461, a selective inhibitor of Pol I transcription [7], was much more effective in cells with wt p53 in com- Melphalan* + - parison with those with mutated p53 [75, 79]. Cisplatin* + - On the other hand, it is worth noting that although Oxaliplatin* + - p53 stabilization appears to be the main mechanism by Cyclophosphamide*+ - which inhibitors of ribosomal biogenesis exert their cy- Intercalating agents: tostatic and cytotoxic action, there is evidence that these Doxorubicin * + - effects can be also caused in a p53-independent way. Depletion of the catalytic subunit of RNA polymerase I Mitoxantrone * + - inhibited the synthesis of rRNA and hindered cell cycle Actinomycin D * + - progression in cells with inactivated p53, as a conse- Mitomycin C + - quence of downregulation of the transcription factor Antimetabolites: E2F-1. Downregulation of E2F-1 was due to release of Methotrexate * + - the ribosomal protein L11, which inactivated the E2F-1- 5-Fluorouracil - + stabilizing function of the E3 ubiquitin protein ligase MDM2 [79]. Furthermore, CX-5461 can induce p53-in- Topoisomerase inhibitors: dependent G2 checkpoint and apoptosis through activa- Camptothecin - + tion of the ataxia telangiectasia mutated (ATM) and Etoposide* - + ataxia telangiectasia and Rad3-related (ATR) kinase Kinase inhibitors: pathway, in the absence of DNA damage [80, 81]. Flavopiridol* - + Regarding hematological malignancies, there is Roscovitine - + evidence that p53 status is an important factor deter- mining the response to currently used chemotherapy Rapamycin + - regimens for lymphoma and leukemia treatment, which Proteasome inhibitors: are based on drugs hindering ribosome biogenesis [5]. Bortezomib* - + Anthracycline-based polychemotherapy represents the Translation inhibitors: standard therapeutic approach for pediatric acute Homoharringtonine* - + lymphoblastic leukemia (ALL) and multiple lymphoma Mitosis inhibitors: subtypes of the adult [including Hodgkin lymphoma (HL), diffuse large B cell lymphoma (DLBCL), and ana- Vinblastine* - + plastic large T cell lymphoma (ALCL)]. More in detail, rRNA polymerase I inhibitors: the ABVD (doxorubicin, bleomycin, vinblastine, and CX-5461*+ - dacarbazine) and the CHOP (cyclophosphamide, doxo- * drugs currently used or in clinical development for the treatment of rubicin, vincristine, and prednisone) regimens represent lymphomas and leukemia Cyclophosphamide is metabolized to acrolein, which is responsible for the the treatments of choice in HL, DLBCL, and ALCL re- inhibition of rRNA transcription [60, 61] spectively. In general, the cure rates of antracycline- CX-5461 is in phase I clinical trial in patients with haematological based regimens have been proved to be variable, being malignancies and in phase I/II trial in patients with breast cancer high for pediatric ALL and Hodgkin lymphoma [82, 83], of chemotherapeutic agents inhibiting ribosome biogen- intermediate for DLBCL [84–86] and ALCL [87, 88], esis should be obviously affected by the status of p53 and low for in indolent B cell lymphoma [89]. Similar [64, 77, 78]. Several lines of preclinical and clinical evi- considerations apply for myeloid disorders where dence support this notion. Indeed, actinomycin D, at a anthracycline-based polychemotherapy has been shown dose that exclusively hinders rDNA transcription, in- to be effective certain forms of acute myeloid leukemia duced a cell cycle arrest with cell accumulation in G1 (reviewed in [90–92]), whereas chronic myeloid neo- and, to a lesser extent, in G2 phase in p53 proficient cell plasms are considered virtually incurable with standard lines [38, 64] whereas these changes in cell cycle distri- polychemotherapy (reviewed in [93]). The intrinsic cur- bution appeared to be reduced if cells were previously si- ability of the aforementioned hematologic cancers relies lenced for p53 expression [64]. The same occurs in cells on precise biological characteristics of cancer cells, and Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 5 of 13 the p53 status has been demonstrated to represent an RB status and its implications on the clinical outcome important prognostic factor. after treatment with drugs inhibiting ribosome biogenesis. In line with this concept, the presence of TP53 genomic In a series of breast cancers treated with an adjuvant che- alterations in DLBCL and chronic lymphoid leukemia is a motherapeutic protocol including cyclophosphamide, well-established negative prognostic predictor [14, 16, 94, methotrexate, and 5-fluorouracil, the presence of a wild- 95]. DLBCL harboring alterations of the p53 pathway are type or mutated p53, considered independently of the RB often nonresponsive to CHOP plus rituximab (R) che- status, proved to have a null prognostic value. However, moimmunotherapy and are characterized by shorter over- by excluding the cases with no pRb expression or all survival. In CLL, patients harboring 17p deletions or inactivated-hyper-phosphorylated pRb, the p53 status re- TP53 mutations are refractory to standard chemotherapy sulted the only factor predicting the patient clinical out- and are currently treated with chemo-free treatments in- come with patients with wt TP53 having a much better cluding inhibitors of B cell receptor signaling or bcl-2 in- prognosis compared to those with mutated TP53.Worth hibitors [96]. In acute myeloid leukemia, the presence of of noting, the lack of pRb expression was the only inde- TP53 mutations is a powerful negative prognostic pre- pendent factor predicting a good clinical outcome in pa- dictor, being associated with refractoriness to current tients treated with adjuvant chemotherapy [101, 102]. anthracycline-based induction therapies [92, 97]. Finally, Moreover, an RB loss gene expression signature was dem- the presence of TP53 gene mutations predicts the out- onstrated to be associated with increased pathological come after induction and reinduction chemotherapy in complete response to neoadjuvant chemotherapy in both acute lymphoid leukemia [98]. estrogen-receptor positive and negative breast cancers The prognostic value of genomic alterations of TP53 [102]. Although the role of pRb pathway has not been has been recently evaluated across a wide variety of evaluated as extensively as p53, similar observations were hematological malignancies confirming the role of the reported in hematological malignancies. In anaplastic p53 axis in determining the efficacy of chemotherapy in large cell lymphoma, absence of pRb expression was this setting [15]. observed in 40% of cases and hyperphosphorylation of pRb was detected in a significant fraction of RB positive The pRb status patients, consistent with RB inactivation. Notably, these These experimental and clinical data indicate that wild- alterations correlated with a favorable clinical outcome type TP53 is a necessary requisite for the activation of [103]. In chronic lymphoid leukemia, 13q14 deletion is the mechanisms leading to cell cycle arrest and/or apop- a frequent genomic alteration, and although the specific totic cell death in cancer cells treated with drugs inhibit- pathogenetic role of RB1 loss in the context of 13q14 ing ribosome biogenesis. deletion is yet to be determined, this cytogenetic abnor- There is evidence that this could be mostly true in the mality predicts good clinical outcome following therapy case of a normally functioning pRb pathway. Indeed, the with the FCR (fludarabine, cyclophosphamide, rituxi- absence of pRb could be a major factor conditioning the mab) regimen [104]. sensitivity of cancer cells to the exposure of RiBi inhibi- Similarly, trisomy 12 (resulting in copy number gain of tors, also when the p53 pathway is dysfunctional [77]. Pre- CDK4 with consequent hyperphosphorylation and in- liminary studies on this topic were conducted on solid activation of pRb) is associated with excellent outcomes tumor models, such as breast cancer. In fact, the contem- following chemoimmunotherapy [104]. Of note, the con- porary absence of pRb and functional p53 has been shown temporary presence of 13q14 deletion seems to attenu- to be responsible for a marked reduction of the cell popu- ate the adverse outcome related to the presence of TP53 lation growth after the inhibition of ribosome biogenesis deletions in CLL [105]. Since the RB1 locus is affected in by actinomycin D, 5-fluorouracyl, methotrexate, and less than 50% of CLL cases harboring 13q14 deletions doxorubicin, which was even greater than that observed [106], it would be interesting to investigate whether spe- in p53 proficient cells [64, 78]. The cause of this increased cific loss of RB1 attenuates the poor prognosis related to sensitivity lies in the complete abrogation of the two cell TP53 alterations. In conclusion, these data taken to- cycle checkpoints in the absence of RB [19, 99, 100]: in gether indicate that (1) the presence of wt p53 associated cells lacking RB, the inhibition of ribosome biogenesis with a normal downstream pRB pathway is an important does not hinder the cell cycle progression, thus leading characteristic which render cancer cells very sensitive to the cells to divide without having reached an appropriate drugs inhibiting ribosome biogenesis and (2) cancer cells ribosome complement. Very rapidly, the reduction of with RB1 loss could be sensitive to ribosome biogenesis ribosome complement becomes incompatible with cell inhibitors irrespective of the p53 status. survival and a progressive increase of apoptotic cell death However, the integrity of the p53/pRb pathway might occurs [64]. These experimental data are consistent with not be the only factor affecting response to ribosomal studies investigating the relationship between the p53 and biogenesis inhibition, as described below. Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 6 of 13 The rate of ribosome biogenesis of the cell Ribosomal protein deletions and mutations Other than arresting cell cycle progression, stabilized Since the main mechanism involved in p53 stabilization p53 may cause programmed cell death by inducing tran- upon ribosome biogenesis inhibition is represented by scription of pro-apoptotic factors [63, 65, 66]. Induction the binding of RPs to MDM2, mutations of ribosomal of apoptosis by inhibitors of ribosome biogenesis de- proteins may constitute another factor influencing the pends on the level of p53 stabilization, apoptosis being response of cancer cells to ribosome biogenesis inhibi- activated only by high amount of stabilized p53. In turn, tors. As reported above, RPL5 and RPL11 play a major the amount of stabilized p53 was shown to be directly role in MDM2 inactivation. However, many other RPs, related to the ribosome biogenesis rate of the cell. This including RPL3, RPL6, RPL23, RPL26, RPL37, RPS7, was demonstrated by using four drugs, which inhibit RPS14, RPS15, RPS19, RPS20, RPS25, RPS26, and rRNA synthesis at different steps: actinomycin D, doxo- RPS27, have been shown to bind to MDM2, thus stabil- rubicin, 5-fluorouracyl, and CX-5461 [63]. In cells char- izing p53 after induction of ribosomal stress (see for a acterized by a high rate of rRNA transcription, the recent and comprehensive review: [52]). There is in- inhibition of ribosome biogenesis caused a significantly creasing evidence for the presence of ribosomal protein greater degree of p53 stabilization and consequent copy number changes and mutations in many types of greater expression of the pro-apoptotic members of the cancer. Regarding the RPs of the large ribosome subunit, Bcl-2 gene family, PUMA, and BAX, compared to those exome sequencing demonstrated the presence of muta- characterized by a lower baseline rRNA synthesis. Ac- tions of RPL5 in T cell acute lymphoblastic leukemia cordingly, apoptotic cell death occurred in cells with a (T-ALL) [108] and in glioblastoma [109], and loss of the high rRNA synthesis and not in cells with a low ribo- 1p22.1 region encompassing the RPL5 gene was found some biogenesis rate, the latter showing only cell cycle in 20% of multiple myeloma cases (MM) [110]. Further- arrest. The tight relationship between the level of p53 more, RPL5 and RPL10 mutations were recently ob- stabilization and the rRNA synthesis rate was due to the served, even though at low frequency, in MM [111]. The fact that, upon ribosome biogenesis inhibition, different frequency of inactivating RPL5 mutations and deletions amounts of RPs, no longer used for ribosome building, was found to be 11% in glioblastoma, 28% in melanoma, bind to MDM2, thus hindering with higher efficiency and 34% in breast cancer patients [112]. In T-ALL, the proteasomal degradation of p53 [63]. Interestingly, in RPL10 and RPL11 mutations have been also described cells with low rRNA synthesis (in which the inhibition of [108, 113] and RPL22 was found to be deleted in about ribosome biogenesis stabilized p53 in a level that was not 10% patients [114]. RPL22 mutations were observed to sufficient for apoptosis induction), the combined treat- occur with high frequency in endometrial [115, 116] and ment with hydroxyurea which activates p53 with a differ- colorectal cancer [117] with microsatellite instability. ent mechanism allowed to increase the total amount of Regarding the proteins constituting the small ribosome stabilized p53 inducing apoptotic cell death [55]. subunit, whole exome sequencing of chronic lympho- Since the induction of cell death, and not cell cycle cytic leukemia showed recurrent mutations of RPS15 arrest, is the main goal of cancer chemotherapy, these ob- [117, 118] while mutations of RPS20 are associated with servations might be relevant for establishing more effect- colorectal carcinoma [119]. There are still few data on ive and appropriate therapeutic protocols. In fact, this the effect of ribosomal protein deletion or mutations on model implies that ribosome biogenesis inhibitors as sin- the response to chemotherapeutic treatments. Experi- gle agents could be highly effective in p53 wild-type can- ments conducted using cancer cell lines demonstrated cers with a high ribosome biogenesis rate, by inducing that silencing the expression of RPL5 and RPL11 apoptotic cell death, whereas for treating cancers with a strongly reduced the stabilization and activation of p53 low ribosome biogenesis rate, they should be combined caused by selective rRNA transcription inhibitors [120, with drugs capable of stabilizing p53 or inducing apoptosis 121], suggesting that cancers carrying these genetic through different mechanisms. This model applies well in changes should be resistant to chemotherapy based on the setting of TP53 wild-type lymphoproliferative neo- inhibitors of ribosome biogenesis. plasms, where aggressive lymphomas such as DLBCLs, Up to now, the only clinical evidence of the impact of characterized by high ribosomal biogenesis rates [107], RP genetic changes on chemotherapy resistance based can be cured with standard R-CHOP polychemotherapy on a reduced activation of the RP-MDM2-p53 pathway [84–86], whereas indolent B cell non-Hodgkin lymph- comes from the study by Ljungström et al. [118] on the omas (such as small lymphocytic lymphoma/chronic relationship between RPS15 mutations and clinical out- lymphoid leukemia, marginal zone lymphoma, and follicu- come of patients with chronic lymphocytic leukemia. lar lymphomas), characterized by low ribosomal biogen- The authors found that patients with RPS15 mutations, esis rates [107], are virtually incurable with the same type but carrying wild-type TP53, treated with standard of polychemotherapy [89]. chemoimmunotherapy (combination of fludarabine, Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 7 of 13 cyclophosphamide, and rituximab), had a shorter 10- adverse outcome in TP53 wild-type patients (manu- year survival compared with patients without mutated script submitted). RPS15, and an overall survival similar to patients charac- In conclusion, although preliminary evidence suggests terized by other adverse-prognostic markers. In the same that RP mutations could provide cancer cells with alter- study, the authors, using a human tumor cell line, dem- native mechanisms to inactivate p53-mediated responses onstrated that transiently expressed mutant RPS15 re- to nucleolar stress, more studies are needed on the oc- duced the expression of p53 due to an increased currence of RP gene deletions and mutations in cancer ubiquitin-mediated p53 degradation in comparison with cells and their influence on p53 stabilization and thera- cells carrying wild-type RPS15. It could be possible that peutic response after treatment with ribosome biogen- mutated RPS15 is not capable of neutralizing the esis inhibitors. MDM2-mediated p53 digestion [122], thus reducing the induction of stabilized p53 upon chemotherapy treat- Mutated nucleophosmin ment. In line with these data, our group recently found Nucleophosmin (NPM1), also called protein B23, numa- non-recurrent mutations of multiple RP genes in a trin, and NO38, is a non-ribosomal phosphoprotein, pri- significant fraction of DLBCL cases (> 10%) and mary located in the nucleolus [123, 124]. NPM1 shuttles RPS12 and RPL22 deletions in up to 20% of cases. between the nucleolus and the cytoplasm [125] and ex- Furthermore, our preliminary data indicate that these erts a series of different biochemical functions, some of alterations are mutually exclusive with TP53 muta- them being independent of ribosome biogenesis (see for tions and that RP mutations could be associated with review [126–129]). Regarding the relationship between Table 2 Overview of genomic alterations involved in the regulation of the RP/MDM2/p53 axis in hematologic malignancies Genomic alteration Disease type Incidence of the alteration Prognostic impact Proposed Mechanism Reference TP53 mutation DLBCL 22%-24% Poor Impaired p53 mediated response [14, 146] to nucleolar stress CLL 7-9% Poor [94, 147–149] ALCL 8% Poor [145] ALL 14-15% Poor [15, 150] AML 5%-9% Poor [92, 151] MM <5% Poor [152] TP53 deletion DLBCL 12% Poor [16] CLL 5-12% Poor [147, 148] ALL 11% Poor [15] MM 9.5% Poor [152] ARF deletion DLBCL 35% Poor Increased MDM2-dependent p53 [153] degradation FL 8% Poor [154] ALL 14-15% Poor [15, 150, 155] RB1 loss DLBCL 11% Neutral Loss of G1/S checkpoint [156] CLL 20% Neutral [157] ALCL 40% Good [103] ALL 9% Neutral [158, 159] RPS15 mutation CLL 19% (RELAPSE) Poor Impaired p53 mediated response [118] to nucleolar stress RPL5 mutation MM Sporadic NE [111] T-ALL <5% NE [108] RPL5 deletion MM 20% Poor [110] RPL10 mutation T-ALL 5% NE [108] RPL22 deletion T-ALL 10% NE [160] NPM1 mutation AML 53% Good* Increased sensitivity to nucleolar stress [91] NPM1-ALK ALCL 55% Good [161] Abbreviations: NE (not evaluated), DLBCL (diffuse large B-cell lymphoma), FL (Follicular lymphoma), CLL (chronic lymphoid leukemia), ALCL (anaplastic large T-cell lymphoma), ALL (acute lymphoid leukemia), T-ALL (T-cell acute lymphoid leukemia), MM (Multiple Myeloma), AML (acute myeloid leukemia) *Associated with good prognosis in the absence of FLT3 genomic alterations Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 8 of 13 NPM1 and ribosome biogenesis, there is evidence that containing anthracyclines and cytarabine [91, 141]. This NPM1 plays a role in rRNA maturation [130] and its is probably due to the fact that leukemic cells with mu- chaperone activity may facilitate the process of ribosome tated NPM1 maintain a functional wild-type p53 [137]. assembly [131]. Furthermore, NPM1 has been shown to In line with this data, a recent study reported that pa- be an important mediator, connecting the BCR-ABL net- tients with AML with mutated NPM1, not eligible for work to ribosome biogenesis and, hence, protein synthe- intensive chemotherapy or with refractory or relapsed sis and cell growth in chronic myelogenous leukemia disease, may be successfully treated with actinomycin D, [132]. Lastly, in proliferating cells, the amount of NPM1 at the same dose as that used for low-risk gestational is directly related to the rRNA transcription rate [133] trophoblastic tumors [142]. The rationale at the basis of and in human cancer cell lines to the nucleolar size and this therapeutic strategy is that leukemic cells with mu- to the rate of cell proliferation [134]. tated NPM1 may have a more vulnerable nucleolus to Quantitative and qualitative changes of NPM1 have the stress induced by the inhibition of ribosome biogen- been reported to occur in many human malignancies esis, resulting in a very strong p53-mediated response. (see for review [126]). Heterozygous NPM1 mutations NPM1 is also a frequent target of chromosomal translo- were observed to occur in about 30% of patients with cations. The NPM1-ALK (anaplastic lymphoma kinase) acute myeloid leukemia (AML) and, with very few ex- fusion protein is the hallmark of ALK-positive anaplastic ceptions, were restricted to exon 12 [135, 136]. Mutant large cell lymphoma (reviewed in [143]). The NPM1- NPM1 is delocalized to the cytoplasm (NPM1c+) while ALK fusion protein activates a series of cellular signaling the amount of wild-type NPM1 located in the nucleolus pathways boosting lymphomagenesis while inhibiting is reduced as a consequence of haploinsufficiency and p53 activity with MDM2 and JNK (c-Jun N-terminal formation of heterodimers with mutated NPM1 in the kinase) dependent mechanisms [144]. Therefore, ALK- cytoplasm [136]. Importantly, NPM1 mutations are positive ALCL often retain a functional p53-mediated re- mutually exclusive with TP53 mutations [137] and con- sponse to nucleolar stress, and accordingly TP53 muta- sistent with this observation the presence of NPM1c+ tions are rare in NPM1-ALK-positive ALCL. In line with inhibits p53-mediated responses: in fact cytoplasmic these findings, NPM1-ALK-positive ALCL are character- NPM1 localization determines sequestration of ARF ized by a better prognosis following conventional CHOP tumor suppressor in the cytoplasm, therefore limiting compared to their ALK negative counterparts. Further the interaction of ARF with MDM2 with consequent in- investigations on the relationship between the functional creased p53 degradation [138–140]. It is noteworthy that state of the nucleolus and the response to ribosome bio- from the clinical point of view acute myeloid leukemia genesis inhibitors should be conducted with the aim of with mutated NPM1 is characterized by a better progno- establishing therapeutic protocols based on selective in- sis due to a higher remission rate after chemotherapy hibition of ribosome biogenesis. Fig. 2 Schematic model representing the relationship between certain intrinsic cancer cell characteristics and curability of hematologic malignancies following chemotherapy based on drugs inhibiting ribosome biogenesis. Cancers with wild-type TP53, high ribosome biogenesis rate, loss of retinoblastoma protein, mutated NPM1 are characterized by good prognosis following chemotherapy (this is the case of TP53 wild-type HL, ALCL, DLBCL, NPM1c+ AML). At the opposite side of the spectrum, cancers characterized by mutant TP53 or mutant ribosomal proteins genes are associated with a low cure rate (certain forms of DLBCL, MM, T-ALL, CLL, AML). In the middle, cancers with low ribosomal biogenesis rate and wild-type TP53 harbor an intermediate cure rate (FL, other indolent B cell lymphoma subtypes) Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 9 of 13 Conclusions Funding This work was supported by the Roberto and Cornelia Pallotti Legacy for Despite the advent of personalized medicine, current Cancer Research. treatment algorithms do not take into account important biological parameters which have been demonstrated to Availability of data and materials Data sharing is not applicable to this article as no datasets were generated affect the cancer response to chemotherapeutic agents or analyzed during the current study. (these factors are summarized in Table 2)[14–16, 91, 92, 94, 103, 108, 110, 111, 118, 146–161]. There is now evi- Authors’ contributions ED conceived the structure of the review and wrote the manuscript; AL dence that the efficacy of many of the chemotherapeutic helped with the manuscript writing; DT conceived the structure of the drugs used for cancer treatment is related to p53 review and wrote the manuscript. All authors read and approved the final stabilization consequent to ribosome biogenesis inhib- manuscript. ition (Fig. 1), and efforts are ongoing to develop new Ethics approval and consent to participate drugs that can selectively target ribosome biogenesis, Not applicable without having the genotoxic effects proper of standard Competing interests chemotherapeutic agents. In this context, it is worth The authors declare that they have no competing interests. mentioning the selective inhibitor of rRNA transcription, the CX-5461 molecule [7, 75], which may represent a Publisher’sNote new, very interesting strategy for cancer therapy [12, Springer Nature remains neutral with regard to jurisdictional claims in 162–164]. 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Treating hematological malignancies with drugs inhibiting ribosome biogenesis: when and why

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Abstract

It is well known that chemotherapy can cure only some cancers in advanced stage, mostly those with an intact p53 pathway. Hematological cancers such as lymphoma and certain forms of leukemia are paradigmatic examples of such scenario. Recent evidence indicates that the efficacy of many of the alkylating and intercalating agents, antimetabolites, topoisomerase, and kinase inhibitors used in cancer therapy is largely due to p53 stabilization and activation consequent to the inhibition of ribosome biogenesis. In this context, innovative drugs specifically hindering ribosome biogenesis showed preclinical activity and are currently in early clinical development in hematological malignancies. The mechanism of p53 stabilization after ribosome biogenesis inhibition is a multistep process, depending on specific factors that can be altered in tumor cells, which can affect the antitumor efficacy of ribosome biogenesis inhibitors (RiBi). In the present review, the basic mechanisms underlying the anticancer activity of RiBi are discussed based on the evidence deriving from available preclinical and clinical studies, with the purpose of defining when and why the treatment with drugs inhibiting ribosomal biogenesis could be highly effective in hematological malignancies. Keywords: Ribosome biogenesis inhibitors, Chemotherapy, Lymphoma, Leukemia, Ribosomal proteins, MDM2, p53, pRb Background selectively hindering the transcription of ribosomal (r) The ribosome biogenesis is defined as the process of RNA, thus inhibiting ribosome biogenesis without hav- building new ribosomes, the intracellular organelles ing genotoxic effects, have been proposed as a new where protein synthesis takes place. therapeutic approach, based on p53 activation [7–12]. In recent years, several studies on the relationship be- However, it is known since long time that chemotherapy tween cell growth and proliferation produced important can cure only some cancers once they reach advanced data regarding the mechanisms linking ribosome biogen- stages. In fact, despite initial responses, the majority of esis, which is at the basis of cell growth, to the progression metastatic solid tumors ultimately progress under chemo- through the cell cycle phases of the proliferating cell. There therapy treatment. Hematological malignancies (such as is now evidence that a perturbed ribosome biogenesis acti- lymphomas and acute leukemias) represent paradigmatic vates a pathway leading to the stabilization and activation examples of the few cancers that can be cured by chemo- of the tumor suppressor protein p53, which in turn induces therapeutic agents and will be the main topic of the cell cycle arrest and/or apoptotic cell death [1–4]. present review [13]. The basic biological characteristic Current evidence indicates that inhibition of ribosome underlying the intrinsic curability of such cancers is that, biogenesis represents a major mechanism by which in a significant fraction of cases, they retain a functional many of the currently used chemotherapeutic drugs p53-mediated response to nucleolar stress arising from (alkylating and intercalating agents, antimetabolites, ribosomal biogenesis inhibition; on the other hand, as a topoisomerase inhibitors) exert their cytotoxic activity matter of fact, the presence of genomic alterations of the on cancer cells [5, 6]. Importantly, a series of new drugs TP53 gene is an established negative prognostic predictor in lymphoma, acute and chronic leukemias treated with chemotherapy regimens [14–17]. * Correspondence: enrico.derenzini@ieo.it; davide.trere@unibo.it European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy DIMES, Università di Bologna, Via Massarenti 9, Bologna, Italy © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 2 of 13 Since p53 stabilization and activation is a multistep binding factor (UBF), are present [33]. For the transcrip- and tightly regulated process, in principle, the prerequis- tion of the 5S rRNA by Pol III, the transcription factors ite for the antitumor efficacy of drugs inhibiting ribo- TFIIIC and TFIIIB are necessary [34–36]. In proliferat- some biogenesis should be the presence in the tumor ing cells, the rate of ribosome biogenesis is enhanced in cells, other than a normally functioning p53, also of order to assure an adequate ribosome complement for those factors necessary for the activation of p53 and the the daughter cells and inhibition of ribosome biogenesis induction of a p53-mediated cell cycle arrest and/or the arrests cell cycle progression [37]. Furthermore, the rate apoptosis. These factors, which control cell cycle pro- of ribosome biogenesis influences the length of the cell gression in normally proliferating cells [18], are qualita- cycle: higher the level of ribosome biogenesis, more tively and quantitatively altered in the large number of rapid the cell cycle progression [38]. Ribosome biogen- cancers [19, 20], thus influencing the sensitivity to ribo- esis rate in cancer shows high variability, depending on a some biogenesis (RiBi) inhibitors. multiplicity of factors including the activation of specific Therefore, it seems timely to critically review the char- intracellular signaling pathways and deregulated activity acteristics of cancer cells which affect their sensitivity to of oncogenes and tumor suppressors. On the other RiBi inhibitors, with the purpose of highlighting those hand, quantitative and qualitative changes in ribosome parameters which render the treatment with these drugs biogenesis have been shown to facilitate neoplastic appropriate or not in hematological malignancies. For transformation. For a detailed description of the the convenience of the reader, the normal process of relationship between ribosome biogenesis and cancer, ribosome biogenesis will be first briefly described. the reader should refer to [39–44]. In hematological malignancies, such as aggressive lymphoproliferative Ribosome biogenesis neoplasms, it is worth mentioning the oncogenic Ribosomes are ribonucleoprotein particles which are lo- cooperation between the MYC oncogene and the cated in the cytoplasm where, either free or membrane- phosphatidyl-inositol-3-kinase (PI3K) signaling pathway bound, are engaged in protein synthesis. Four types of [45], which converge in stimulating rRNA synthesis and ribosomal RNA (rRNA) molecules and about 80 differ- ribosome biogenesis [46]. ent ribosomal proteins constitute the ribosome. Ribo- some formation occurs mainly in the nucleolus, being Inhibition of ribosome biogenesis activates the later completed in the nucleoplasm and in the cytoplasm RPs/MDM2/p53 pathway (see for reviews: [21–24]). In the nucleolus, ribosomal Available data indicate that the levels of p53 expression genes are transcribed by RNA polymerase I (Pol I) to and activity are mainly regulated by interactions with generate the 47S rRNA precursor, which undergoes to the tumor suppressor MDM2 (murine double minute 2, site-specific methylation and pseudo uridylation, and and HDM2 in humans). MDM2 is an E3 ubiquitin ligase processing to give rise to the mature 18S, 5.8S, and 28S which negatively controls p53 activity in two ways: by rRNA. The fourth types of rRNA, the 5S rRNA, is syn- binding to the protein and inhibiting its transactivation thesized in the nucleoplasm by RNA polymerase III (Pol activity, and by facilitating its proteasome degradation III) and then imported in the nucleolus together with [47–49]. In normal proliferating cells, the level of p53 is the ribosomal proteins (RPs), whose mRNA is tran- maintained low because of the binding with MDM2 with scribed by RNA polymerase II (Pol II). The assembling of consequent p53 ubiquitination and proteasome digestion rRNA molecules with the RPs constitutes the two sub- [50]. When a perturbation in the ribosome biogenesis units of the mature ribosome, the large 60S and the small occurs (ribosome stress), it results in the binding of sev- 40S subunit. The large 60S subunit is constituted by one eral ribosomal proteins, no longer used for ribosome each of the 28S, 5.8S, and 5S RNA molecules, together building, to MDM2. This binding relieves the inhibitory with 47 ribosomal proteins (RPLs); the small 40S subunit activity of MDM2 toward p53 (see reviews [2–4, 51, 52]) contains only one 18S RNA molecule and 33 ribosomal (Fig. 1). Although there is evidence that RPL5, RPL11, proteins (RPSs) [25, 26]. Both subunits migrate from the and RPL23 play a major role in neutralization of MDM2 nucleolus to the cytoplasm where they form the 80S ribo- activity and in the induction of p53 stabilization [50, 53– some particle. In the process of ribosome biogenesis, more 58], the list of ribosomal proteins (of both large and than 150 non-ribosomal proteins and around 70 small nu- small ribosomal subunit) able to inhibit MDM2 activity cleolar RNAs are involved [27–32]. and to stabilize p53 upon “ribosomal stress” is rapidly For the transcription of the of 47S pre-rRNA, the as- expanding [52]. For a valid binding to MDM2 and its in- sembly of a specific multiprotein complex at the rDNA activation, the RPL11 and RPL5 must form a complex promoter containing Pol I is required. In this complex, with the 5S rRNA and all the components of this com- three basal factors, termed transcription initiation factor plex are necessary for its inhibitory function [59, 60]. I (TIF-I) A, selectivity factor 1 (SL1), and upstream p53 stabilization always causes cell cycle arrest in Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 3 of 13 MDM2 phosphorylation results in increased stability of MDM2 with consequent p53 degradation [69, 70]. As mentioned before, constitutive PI3K signaling is common in lymphoproliferative neoplasms, and PI3K inhibitors are in clinical development in lymphoid cancers. These no- tions could be relevant for designing therapeutic combin- ation strategies aimed at increasing the p53-mediated response to the inhibition of ribosome biogenesis. Development of selective inhibitors of ribosome biogenesis As briefly mentioned before, a strong contribution to p53 activation induced by chemotherapeutic agents is due to the inhibition of ribosomal biogenesis. As re- ported by Burger et al. [5], a series of drugs currently used for treating solid cancers and hematological malig- nancies inhibit ribosome biogenesis at the level of rRNA transcription and/or at the level of rRNA processing (Table 1). To this list, cyclophosphamide and mycophe- Fig. 1 Schematic representation of the pathway activated by drug- nolic acid should be added. Cyclophosphamide, a widely induced perturbation of rRNA synthesis. Ribosomal proteins (RPs), no used anticancer drug, also inhibits rRNA transcription longer used for ribosome building, bind to MDM2, thus inhibiting its [71], after being converted to acrolein [72, 73], and the ubiquitin ligase activity toward p53 and the proteasome digestion of immunosuppressant mycophenolic acid has been dem- the tumor suppressor. As a consequence, p53 accumulates and induces onstrated to inhibit the synthesis of rRNA [74]. transcription of p21, PUMA, and BAX. P21 is responsible for the cell cycle arrest by hindering pRb phosphorylation: in fact, hypo-phosphorylated In recent years, several efforts have been made to de- pRb binds to and inhibits the activity of the transcription factor E2F1, velop specific inhibitors of ribosomal biogenesis, in order whose target gene products are necessary for cell cycle progression. to achieve a selective inhibition of rRNA synthesis without The induction of the pro-apoptotic factors PUMA and BAX activates the the genotoxic effects proper of chemotherapeutic drugs. process of apoptotic cell death In this light, it appears to be of particular relevance the CX-5461 molecule which selectively inhibits ribosome proliferating cells and, depending on the quantitative biogenesis, most likely by disrupting the SL-1/rDNA com- level of stabilized p53, also apoptotic cell death [61–63]. plex, promoting a cancer-specific activation of p53. Recent p53 arrests cell cycle progression by inhibiting the phos- preclinical data indicate high activity of CX-5461 in MYC- phorylation of the tumor suppressor retinoblastoma driven lymphoma, providing the rationale for further protein, pRb. In its hypo-phosphorylated form, pRb clinical development of this compound [7, 75, 76]. binds to and inhibits the activity of E2F1, a transcription CX-5361 is currently under phase I clinical trial for the factor whose target genes are necessary for cell cycle treatment of patients with advanced hematologic malig- progression. The inhibition of E2F1 activity by hypo- nancies, including acute myeloid leukemia. phosphorylated pRb reduces the expression of both Finally, there is experimental evidence that a small mo- cyclin E and A, necessary factors for cell cycle progres- lecular compound, BMH-21, and a small-molecule pep- sion from G1 to S phase and from G2 to M phase tide (22mer) also selectively inhibit rDNA transcription. respectively, with consequent cell accumulation in G1 BMH21 binds to GC-rich sequences and inhibits RNA and G2 phase [64]. The induction of apoptotic cell death Pol I activity [9]. It also induces the proteasome- by p53 is a consequence of induced expression of the dependent destruction of the large catalytic subunit in the pro-apoptotic members of the B cell lymphoma 2 (Bcl-2) Pol I complex, as do three other small molecular com- gene family, PUMA, and BAX [63, 65–67](Fig. 1). Finally, pounds, BMH-9, BMH-22, and BMH-23 [10]. The 22mer it should be noted that additional factors may interact targets the interface between RNA polymerase I and Rrn3, with the RPs/MDM2/p53 axis, such as the ARF tumor thus selectively inhibiting the synthesis of rRNA [11]. suppressor and the activation of the PI3K pathway. In fact, ARF loss is a common genetic event in cancer and espe- Factors determining cancer cell sensitivity to drugs cially in aggressive lymphoid neoplasms, resulting in in- inhibiting ribosome biogenesis creased MDM2 activity and increased p53 degradation The p53 status (reviewed in [68]). On the other hand, MDM2 is a down- Since a major effect of ribosome biogenesis inhibition is stream target of the PI3K-AKT axis, and AKT-induced the activation of p53, the cytostatic and cytotoxic effects Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 4 of 13 Table 1 Drugs used to treat hematological and solid with inactivated p53 in which the synthesis of rRNA was malignancies which are effective or highly effective in the hindered by polymerase I silencing [79]. Also, p53 silen- inhibition of rRNA transcription or processing (modified from cing significantly reduced the antiproliferative effects of Burger et al., 2010) [5] 5-fluorouracil and methotrexate or doxorubicin, in human inibition of rRNA synthesis cancer cell lines harboring wild type (wt) p53 [78]and transcription processing treatment of human leukemia and lymphoma cell lines Alkylating agents: with CX-5461, a selective inhibitor of Pol I transcription [7], was much more effective in cells with wt p53 in com- Melphalan* + - parison with those with mutated p53 [75, 79]. Cisplatin* + - On the other hand, it is worth noting that although Oxaliplatin* + - p53 stabilization appears to be the main mechanism by Cyclophosphamide*+ - which inhibitors of ribosomal biogenesis exert their cy- Intercalating agents: tostatic and cytotoxic action, there is evidence that these Doxorubicin * + - effects can be also caused in a p53-independent way. Depletion of the catalytic subunit of RNA polymerase I Mitoxantrone * + - inhibited the synthesis of rRNA and hindered cell cycle Actinomycin D * + - progression in cells with inactivated p53, as a conse- Mitomycin C + - quence of downregulation of the transcription factor Antimetabolites: E2F-1. Downregulation of E2F-1 was due to release of Methotrexate * + - the ribosomal protein L11, which inactivated the E2F-1- 5-Fluorouracil - + stabilizing function of the E3 ubiquitin protein ligase MDM2 [79]. Furthermore, CX-5461 can induce p53-in- Topoisomerase inhibitors: dependent G2 checkpoint and apoptosis through activa- Camptothecin - + tion of the ataxia telangiectasia mutated (ATM) and Etoposide* - + ataxia telangiectasia and Rad3-related (ATR) kinase Kinase inhibitors: pathway, in the absence of DNA damage [80, 81]. Flavopiridol* - + Regarding hematological malignancies, there is Roscovitine - + evidence that p53 status is an important factor deter- mining the response to currently used chemotherapy Rapamycin + - regimens for lymphoma and leukemia treatment, which Proteasome inhibitors: are based on drugs hindering ribosome biogenesis [5]. Bortezomib* - + Anthracycline-based polychemotherapy represents the Translation inhibitors: standard therapeutic approach for pediatric acute Homoharringtonine* - + lymphoblastic leukemia (ALL) and multiple lymphoma Mitosis inhibitors: subtypes of the adult [including Hodgkin lymphoma (HL), diffuse large B cell lymphoma (DLBCL), and ana- Vinblastine* - + plastic large T cell lymphoma (ALCL)]. More in detail, rRNA polymerase I inhibitors: the ABVD (doxorubicin, bleomycin, vinblastine, and CX-5461*+ - dacarbazine) and the CHOP (cyclophosphamide, doxo- * drugs currently used or in clinical development for the treatment of rubicin, vincristine, and prednisone) regimens represent lymphomas and leukemia Cyclophosphamide is metabolized to acrolein, which is responsible for the the treatments of choice in HL, DLBCL, and ALCL re- inhibition of rRNA transcription [60, 61] spectively. In general, the cure rates of antracycline- CX-5461 is in phase I clinical trial in patients with haematological based regimens have been proved to be variable, being malignancies and in phase I/II trial in patients with breast cancer high for pediatric ALL and Hodgkin lymphoma [82, 83], of chemotherapeutic agents inhibiting ribosome biogen- intermediate for DLBCL [84–86] and ALCL [87, 88], esis should be obviously affected by the status of p53 and low for in indolent B cell lymphoma [89]. Similar [64, 77, 78]. Several lines of preclinical and clinical evi- considerations apply for myeloid disorders where dence support this notion. Indeed, actinomycin D, at a anthracycline-based polychemotherapy has been shown dose that exclusively hinders rDNA transcription, in- to be effective certain forms of acute myeloid leukemia duced a cell cycle arrest with cell accumulation in G1 (reviewed in [90–92]), whereas chronic myeloid neo- and, to a lesser extent, in G2 phase in p53 proficient cell plasms are considered virtually incurable with standard lines [38, 64] whereas these changes in cell cycle distri- polychemotherapy (reviewed in [93]). The intrinsic cur- bution appeared to be reduced if cells were previously si- ability of the aforementioned hematologic cancers relies lenced for p53 expression [64]. The same occurs in cells on precise biological characteristics of cancer cells, and Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 5 of 13 the p53 status has been demonstrated to represent an RB status and its implications on the clinical outcome important prognostic factor. after treatment with drugs inhibiting ribosome biogenesis. In line with this concept, the presence of TP53 genomic In a series of breast cancers treated with an adjuvant che- alterations in DLBCL and chronic lymphoid leukemia is a motherapeutic protocol including cyclophosphamide, well-established negative prognostic predictor [14, 16, 94, methotrexate, and 5-fluorouracil, the presence of a wild- 95]. DLBCL harboring alterations of the p53 pathway are type or mutated p53, considered independently of the RB often nonresponsive to CHOP plus rituximab (R) che- status, proved to have a null prognostic value. However, moimmunotherapy and are characterized by shorter over- by excluding the cases with no pRb expression or all survival. In CLL, patients harboring 17p deletions or inactivated-hyper-phosphorylated pRb, the p53 status re- TP53 mutations are refractory to standard chemotherapy sulted the only factor predicting the patient clinical out- and are currently treated with chemo-free treatments in- come with patients with wt TP53 having a much better cluding inhibitors of B cell receptor signaling or bcl-2 in- prognosis compared to those with mutated TP53.Worth hibitors [96]. In acute myeloid leukemia, the presence of of noting, the lack of pRb expression was the only inde- TP53 mutations is a powerful negative prognostic pre- pendent factor predicting a good clinical outcome in pa- dictor, being associated with refractoriness to current tients treated with adjuvant chemotherapy [101, 102]. anthracycline-based induction therapies [92, 97]. Finally, Moreover, an RB loss gene expression signature was dem- the presence of TP53 gene mutations predicts the out- onstrated to be associated with increased pathological come after induction and reinduction chemotherapy in complete response to neoadjuvant chemotherapy in both acute lymphoid leukemia [98]. estrogen-receptor positive and negative breast cancers The prognostic value of genomic alterations of TP53 [102]. Although the role of pRb pathway has not been has been recently evaluated across a wide variety of evaluated as extensively as p53, similar observations were hematological malignancies confirming the role of the reported in hematological malignancies. In anaplastic p53 axis in determining the efficacy of chemotherapy in large cell lymphoma, absence of pRb expression was this setting [15]. observed in 40% of cases and hyperphosphorylation of pRb was detected in a significant fraction of RB positive The pRb status patients, consistent with RB inactivation. Notably, these These experimental and clinical data indicate that wild- alterations correlated with a favorable clinical outcome type TP53 is a necessary requisite for the activation of [103]. In chronic lymphoid leukemia, 13q14 deletion is the mechanisms leading to cell cycle arrest and/or apop- a frequent genomic alteration, and although the specific totic cell death in cancer cells treated with drugs inhibit- pathogenetic role of RB1 loss in the context of 13q14 ing ribosome biogenesis. deletion is yet to be determined, this cytogenetic abnor- There is evidence that this could be mostly true in the mality predicts good clinical outcome following therapy case of a normally functioning pRb pathway. Indeed, the with the FCR (fludarabine, cyclophosphamide, rituxi- absence of pRb could be a major factor conditioning the mab) regimen [104]. sensitivity of cancer cells to the exposure of RiBi inhibi- Similarly, trisomy 12 (resulting in copy number gain of tors, also when the p53 pathway is dysfunctional [77]. Pre- CDK4 with consequent hyperphosphorylation and in- liminary studies on this topic were conducted on solid activation of pRb) is associated with excellent outcomes tumor models, such as breast cancer. In fact, the contem- following chemoimmunotherapy [104]. Of note, the con- porary absence of pRb and functional p53 has been shown temporary presence of 13q14 deletion seems to attenu- to be responsible for a marked reduction of the cell popu- ate the adverse outcome related to the presence of TP53 lation growth after the inhibition of ribosome biogenesis deletions in CLL [105]. Since the RB1 locus is affected in by actinomycin D, 5-fluorouracyl, methotrexate, and less than 50% of CLL cases harboring 13q14 deletions doxorubicin, which was even greater than that observed [106], it would be interesting to investigate whether spe- in p53 proficient cells [64, 78]. The cause of this increased cific loss of RB1 attenuates the poor prognosis related to sensitivity lies in the complete abrogation of the two cell TP53 alterations. In conclusion, these data taken to- cycle checkpoints in the absence of RB [19, 99, 100]: in gether indicate that (1) the presence of wt p53 associated cells lacking RB, the inhibition of ribosome biogenesis with a normal downstream pRB pathway is an important does not hinder the cell cycle progression, thus leading characteristic which render cancer cells very sensitive to the cells to divide without having reached an appropriate drugs inhibiting ribosome biogenesis and (2) cancer cells ribosome complement. Very rapidly, the reduction of with RB1 loss could be sensitive to ribosome biogenesis ribosome complement becomes incompatible with cell inhibitors irrespective of the p53 status. survival and a progressive increase of apoptotic cell death However, the integrity of the p53/pRb pathway might occurs [64]. These experimental data are consistent with not be the only factor affecting response to ribosomal studies investigating the relationship between the p53 and biogenesis inhibition, as described below. Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 6 of 13 The rate of ribosome biogenesis of the cell Ribosomal protein deletions and mutations Other than arresting cell cycle progression, stabilized Since the main mechanism involved in p53 stabilization p53 may cause programmed cell death by inducing tran- upon ribosome biogenesis inhibition is represented by scription of pro-apoptotic factors [63, 65, 66]. Induction the binding of RPs to MDM2, mutations of ribosomal of apoptosis by inhibitors of ribosome biogenesis de- proteins may constitute another factor influencing the pends on the level of p53 stabilization, apoptosis being response of cancer cells to ribosome biogenesis inhibi- activated only by high amount of stabilized p53. In turn, tors. As reported above, RPL5 and RPL11 play a major the amount of stabilized p53 was shown to be directly role in MDM2 inactivation. However, many other RPs, related to the ribosome biogenesis rate of the cell. This including RPL3, RPL6, RPL23, RPL26, RPL37, RPS7, was demonstrated by using four drugs, which inhibit RPS14, RPS15, RPS19, RPS20, RPS25, RPS26, and rRNA synthesis at different steps: actinomycin D, doxo- RPS27, have been shown to bind to MDM2, thus stabil- rubicin, 5-fluorouracyl, and CX-5461 [63]. In cells char- izing p53 after induction of ribosomal stress (see for a acterized by a high rate of rRNA transcription, the recent and comprehensive review: [52]). There is in- inhibition of ribosome biogenesis caused a significantly creasing evidence for the presence of ribosomal protein greater degree of p53 stabilization and consequent copy number changes and mutations in many types of greater expression of the pro-apoptotic members of the cancer. Regarding the RPs of the large ribosome subunit, Bcl-2 gene family, PUMA, and BAX, compared to those exome sequencing demonstrated the presence of muta- characterized by a lower baseline rRNA synthesis. Ac- tions of RPL5 in T cell acute lymphoblastic leukemia cordingly, apoptotic cell death occurred in cells with a (T-ALL) [108] and in glioblastoma [109], and loss of the high rRNA synthesis and not in cells with a low ribo- 1p22.1 region encompassing the RPL5 gene was found some biogenesis rate, the latter showing only cell cycle in 20% of multiple myeloma cases (MM) [110]. Further- arrest. The tight relationship between the level of p53 more, RPL5 and RPL10 mutations were recently ob- stabilization and the rRNA synthesis rate was due to the served, even though at low frequency, in MM [111]. The fact that, upon ribosome biogenesis inhibition, different frequency of inactivating RPL5 mutations and deletions amounts of RPs, no longer used for ribosome building, was found to be 11% in glioblastoma, 28% in melanoma, bind to MDM2, thus hindering with higher efficiency and 34% in breast cancer patients [112]. In T-ALL, the proteasomal degradation of p53 [63]. Interestingly, in RPL10 and RPL11 mutations have been also described cells with low rRNA synthesis (in which the inhibition of [108, 113] and RPL22 was found to be deleted in about ribosome biogenesis stabilized p53 in a level that was not 10% patients [114]. RPL22 mutations were observed to sufficient for apoptosis induction), the combined treat- occur with high frequency in endometrial [115, 116] and ment with hydroxyurea which activates p53 with a differ- colorectal cancer [117] with microsatellite instability. ent mechanism allowed to increase the total amount of Regarding the proteins constituting the small ribosome stabilized p53 inducing apoptotic cell death [55]. subunit, whole exome sequencing of chronic lympho- Since the induction of cell death, and not cell cycle cytic leukemia showed recurrent mutations of RPS15 arrest, is the main goal of cancer chemotherapy, these ob- [117, 118] while mutations of RPS20 are associated with servations might be relevant for establishing more effect- colorectal carcinoma [119]. There are still few data on ive and appropriate therapeutic protocols. In fact, this the effect of ribosomal protein deletion or mutations on model implies that ribosome biogenesis inhibitors as sin- the response to chemotherapeutic treatments. Experi- gle agents could be highly effective in p53 wild-type can- ments conducted using cancer cell lines demonstrated cers with a high ribosome biogenesis rate, by inducing that silencing the expression of RPL5 and RPL11 apoptotic cell death, whereas for treating cancers with a strongly reduced the stabilization and activation of p53 low ribosome biogenesis rate, they should be combined caused by selective rRNA transcription inhibitors [120, with drugs capable of stabilizing p53 or inducing apoptosis 121], suggesting that cancers carrying these genetic through different mechanisms. This model applies well in changes should be resistant to chemotherapy based on the setting of TP53 wild-type lymphoproliferative neo- inhibitors of ribosome biogenesis. plasms, where aggressive lymphomas such as DLBCLs, Up to now, the only clinical evidence of the impact of characterized by high ribosomal biogenesis rates [107], RP genetic changes on chemotherapy resistance based can be cured with standard R-CHOP polychemotherapy on a reduced activation of the RP-MDM2-p53 pathway [84–86], whereas indolent B cell non-Hodgkin lymph- comes from the study by Ljungström et al. [118] on the omas (such as small lymphocytic lymphoma/chronic relationship between RPS15 mutations and clinical out- lymphoid leukemia, marginal zone lymphoma, and follicu- come of patients with chronic lymphocytic leukemia. lar lymphomas), characterized by low ribosomal biogen- The authors found that patients with RPS15 mutations, esis rates [107], are virtually incurable with the same type but carrying wild-type TP53, treated with standard of polychemotherapy [89]. chemoimmunotherapy (combination of fludarabine, Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 7 of 13 cyclophosphamide, and rituximab), had a shorter 10- adverse outcome in TP53 wild-type patients (manu- year survival compared with patients without mutated script submitted). RPS15, and an overall survival similar to patients charac- In conclusion, although preliminary evidence suggests terized by other adverse-prognostic markers. In the same that RP mutations could provide cancer cells with alter- study, the authors, using a human tumor cell line, dem- native mechanisms to inactivate p53-mediated responses onstrated that transiently expressed mutant RPS15 re- to nucleolar stress, more studies are needed on the oc- duced the expression of p53 due to an increased currence of RP gene deletions and mutations in cancer ubiquitin-mediated p53 degradation in comparison with cells and their influence on p53 stabilization and thera- cells carrying wild-type RPS15. It could be possible that peutic response after treatment with ribosome biogen- mutated RPS15 is not capable of neutralizing the esis inhibitors. MDM2-mediated p53 digestion [122], thus reducing the induction of stabilized p53 upon chemotherapy treat- Mutated nucleophosmin ment. In line with these data, our group recently found Nucleophosmin (NPM1), also called protein B23, numa- non-recurrent mutations of multiple RP genes in a trin, and NO38, is a non-ribosomal phosphoprotein, pri- significant fraction of DLBCL cases (> 10%) and mary located in the nucleolus [123, 124]. NPM1 shuttles RPS12 and RPL22 deletions in up to 20% of cases. between the nucleolus and the cytoplasm [125] and ex- Furthermore, our preliminary data indicate that these erts a series of different biochemical functions, some of alterations are mutually exclusive with TP53 muta- them being independent of ribosome biogenesis (see for tions and that RP mutations could be associated with review [126–129]). Regarding the relationship between Table 2 Overview of genomic alterations involved in the regulation of the RP/MDM2/p53 axis in hematologic malignancies Genomic alteration Disease type Incidence of the alteration Prognostic impact Proposed Mechanism Reference TP53 mutation DLBCL 22%-24% Poor Impaired p53 mediated response [14, 146] to nucleolar stress CLL 7-9% Poor [94, 147–149] ALCL 8% Poor [145] ALL 14-15% Poor [15, 150] AML 5%-9% Poor [92, 151] MM <5% Poor [152] TP53 deletion DLBCL 12% Poor [16] CLL 5-12% Poor [147, 148] ALL 11% Poor [15] MM 9.5% Poor [152] ARF deletion DLBCL 35% Poor Increased MDM2-dependent p53 [153] degradation FL 8% Poor [154] ALL 14-15% Poor [15, 150, 155] RB1 loss DLBCL 11% Neutral Loss of G1/S checkpoint [156] CLL 20% Neutral [157] ALCL 40% Good [103] ALL 9% Neutral [158, 159] RPS15 mutation CLL 19% (RELAPSE) Poor Impaired p53 mediated response [118] to nucleolar stress RPL5 mutation MM Sporadic NE [111] T-ALL <5% NE [108] RPL5 deletion MM 20% Poor [110] RPL10 mutation T-ALL 5% NE [108] RPL22 deletion T-ALL 10% NE [160] NPM1 mutation AML 53% Good* Increased sensitivity to nucleolar stress [91] NPM1-ALK ALCL 55% Good [161] Abbreviations: NE (not evaluated), DLBCL (diffuse large B-cell lymphoma), FL (Follicular lymphoma), CLL (chronic lymphoid leukemia), ALCL (anaplastic large T-cell lymphoma), ALL (acute lymphoid leukemia), T-ALL (T-cell acute lymphoid leukemia), MM (Multiple Myeloma), AML (acute myeloid leukemia) *Associated with good prognosis in the absence of FLT3 genomic alterations Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 8 of 13 NPM1 and ribosome biogenesis, there is evidence that containing anthracyclines and cytarabine [91, 141]. This NPM1 plays a role in rRNA maturation [130] and its is probably due to the fact that leukemic cells with mu- chaperone activity may facilitate the process of ribosome tated NPM1 maintain a functional wild-type p53 [137]. assembly [131]. Furthermore, NPM1 has been shown to In line with this data, a recent study reported that pa- be an important mediator, connecting the BCR-ABL net- tients with AML with mutated NPM1, not eligible for work to ribosome biogenesis and, hence, protein synthe- intensive chemotherapy or with refractory or relapsed sis and cell growth in chronic myelogenous leukemia disease, may be successfully treated with actinomycin D, [132]. Lastly, in proliferating cells, the amount of NPM1 at the same dose as that used for low-risk gestational is directly related to the rRNA transcription rate [133] trophoblastic tumors [142]. The rationale at the basis of and in human cancer cell lines to the nucleolar size and this therapeutic strategy is that leukemic cells with mu- to the rate of cell proliferation [134]. tated NPM1 may have a more vulnerable nucleolus to Quantitative and qualitative changes of NPM1 have the stress induced by the inhibition of ribosome biogen- been reported to occur in many human malignancies esis, resulting in a very strong p53-mediated response. (see for review [126]). Heterozygous NPM1 mutations NPM1 is also a frequent target of chromosomal translo- were observed to occur in about 30% of patients with cations. The NPM1-ALK (anaplastic lymphoma kinase) acute myeloid leukemia (AML) and, with very few ex- fusion protein is the hallmark of ALK-positive anaplastic ceptions, were restricted to exon 12 [135, 136]. Mutant large cell lymphoma (reviewed in [143]). The NPM1- NPM1 is delocalized to the cytoplasm (NPM1c+) while ALK fusion protein activates a series of cellular signaling the amount of wild-type NPM1 located in the nucleolus pathways boosting lymphomagenesis while inhibiting is reduced as a consequence of haploinsufficiency and p53 activity with MDM2 and JNK (c-Jun N-terminal formation of heterodimers with mutated NPM1 in the kinase) dependent mechanisms [144]. Therefore, ALK- cytoplasm [136]. Importantly, NPM1 mutations are positive ALCL often retain a functional p53-mediated re- mutually exclusive with TP53 mutations [137] and con- sponse to nucleolar stress, and accordingly TP53 muta- sistent with this observation the presence of NPM1c+ tions are rare in NPM1-ALK-positive ALCL. In line with inhibits p53-mediated responses: in fact cytoplasmic these findings, NPM1-ALK-positive ALCL are character- NPM1 localization determines sequestration of ARF ized by a better prognosis following conventional CHOP tumor suppressor in the cytoplasm, therefore limiting compared to their ALK negative counterparts. Further the interaction of ARF with MDM2 with consequent in- investigations on the relationship between the functional creased p53 degradation [138–140]. It is noteworthy that state of the nucleolus and the response to ribosome bio- from the clinical point of view acute myeloid leukemia genesis inhibitors should be conducted with the aim of with mutated NPM1 is characterized by a better progno- establishing therapeutic protocols based on selective in- sis due to a higher remission rate after chemotherapy hibition of ribosome biogenesis. Fig. 2 Schematic model representing the relationship between certain intrinsic cancer cell characteristics and curability of hematologic malignancies following chemotherapy based on drugs inhibiting ribosome biogenesis. Cancers with wild-type TP53, high ribosome biogenesis rate, loss of retinoblastoma protein, mutated NPM1 are characterized by good prognosis following chemotherapy (this is the case of TP53 wild-type HL, ALCL, DLBCL, NPM1c+ AML). At the opposite side of the spectrum, cancers characterized by mutant TP53 or mutant ribosomal proteins genes are associated with a low cure rate (certain forms of DLBCL, MM, T-ALL, CLL, AML). In the middle, cancers with low ribosomal biogenesis rate and wild-type TP53 harbor an intermediate cure rate (FL, other indolent B cell lymphoma subtypes) Derenzini et al. Journal of Hematology & Oncology (2018) 11:75 Page 9 of 13 Conclusions Funding This work was supported by the Roberto and Cornelia Pallotti Legacy for Despite the advent of personalized medicine, current Cancer Research. treatment algorithms do not take into account important biological parameters which have been demonstrated to Availability of data and materials Data sharing is not applicable to this article as no datasets were generated affect the cancer response to chemotherapeutic agents or analyzed during the current study. (these factors are summarized in Table 2)[14–16, 91, 92, 94, 103, 108, 110, 111, 118, 146–161]. There is now evi- Authors’ contributions ED conceived the structure of the review and wrote the manuscript; AL dence that the efficacy of many of the chemotherapeutic helped with the manuscript writing; DT conceived the structure of the drugs used for cancer treatment is related to p53 review and wrote the manuscript. All authors read and approved the final stabilization consequent to ribosome biogenesis inhib- manuscript. ition (Fig. 1), and efforts are ongoing to develop new Ethics approval and consent to participate drugs that can selectively target ribosome biogenesis, Not applicable without having the genotoxic effects proper of standard Competing interests chemotherapeutic agents. In this context, it is worth The authors declare that they have no competing interests. mentioning the selective inhibitor of rRNA transcription, the CX-5461 molecule [7, 75], which may represent a Publisher’sNote new, very interesting strategy for cancer therapy [12, Springer Nature remains neutral with regard to jurisdictional claims in 162–164]. 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Journal

Journal of Hematology & OncologySpringer Journals

Published: May 31, 2018

References

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