RNA-targeted therapeutics in cancer clinical trials: Current status and future directions

Cancer Treatment Reviews 50 (2016) 35–47

Contents lists available at ScienceDirect

Cancer Treatment Reviews journal homepage: www.elsevierhealth.com/journals/ctrv

New Drugs

RNA-targeted therapeutics in cancer clinical trials: Current status and future directions Pedro Barata a, Anil K. Sood b,c,d, David S. Hong e,⇑ a

Department of Solid Tumors, Taussig Cancer Institute, Cleveland Clinic, Cleveland, USA Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA c Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA d Center for RNA Interference and Non-Coding RNA, The University of Texas MD Anderson Cancer Center, Houston, TX, USA e Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA b

a r t i c l e

i n f o

Article history: Received 7 July 2016 Accepted 12 August 2016

Keywords: Cancer clinical trials RNA therapeutics Antisense oligonucleotides miRNA siRNA

a b s t r a c t Recent advances in RNA delivery and target selection provide unprecedented opportunities for cancer treatment, especially for cancers that are particularly hard to treat with existing drugs. Small interfering RNAs, microRNAs, and antisense oligonucleotides are the most widely used strategies for silencing gene expression. In this review, we summarize how these approaches were used to develop drugs targeting RNA in human cells. Then, we review the current state of clinical trials of these agents for different types of cancer and outcomes from published data. Finally, we discuss lessons learned from completed studies and future directions for this class of drugs. Ó 2016 Elsevier Ltd. All rights reserved.

Introduction The ability of RNA interference (RNAi) to silence target genes with high efficiency and specificity has stimulated efforts to develop these molecules into therapeutic agents. This approach is especially compelling within oncology, given that many important targets have proven to be undruggable [1]. RNA-based therapeutics offer a multitude of opportunities, and here we focus on RNA-based therapies that have reached cancer-related clinical trials [2–4]. In this review, we provide an update on current clinical trials of RNA-based therapies for various types of cancer and summarize the outcomes from published data. RNAi cellular processes The cellular process of RNAi occurs in almost all eukaryotic organisms, as described by Ambros [5] and Fire and Mello [6–8]. A long, double-stranded RNA effectively silences the expression of a gene by inducing the cleavage and degradation of a ⇑ Corresponding author at: Department of Investigational Cancer Therapeutics, Clinical Center for Targeted Therapy, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Unit 455, PO Box 301402, Houston, TX 772301402, USA. Room FC8.3050, 1400 Holcombe Blvd, Houston, TX 77030-3722, USA. E-mail address: [email protected] (D.S. Hong). http://dx.doi.org/10.1016/j.ctrv.2016.08.004 0305-7372/Ó 2016 Elsevier Ltd. All rights reserved.

homologous host mRNA [9]. This transcriptional regulation mechanism is also known as gene silencing [10–12]. Since RNAi was discovered, the mechanisms have become apparent. These involve short interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs), and others (Fig. 1). Both siRNA and miRNA, after being processed by the ribonuclease III-like DICER enzyme, interact with RNA-induced silencing complex to block and neutralize the target mRNA [11,13]. A single miRNA can target several mRNAs and a single mRNA can contain several signals for miRNA recognition [14]. Additionally, miRNAs can act as ‘‘replacement therapy” by restoring loss of function, and the effect of miRNAs on cancer cells is being tested in clinical trials, as described below [15]. Synthetic DNA/RNA-like oligonucleotides, known as ASOs, bind to RNA through sequence-specific Watson– Crick base pair interactions [16]. After crossing the cell membrane, ASOs target mRNA directly, in the nucleus or cytosol, thus blocking and neutralizing the targeted miRNA, with the help of the enzyme RNase H (Fig. 1).

Literature search We reviewed published phase I/II/III clinical trials of any form of RNA therapeutics for any human cancer at any stage. We searched for these published articles in the Medline, Embase, Clinicaltrials. com, and Molecularmatch.com electronic databases, as well as

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P. Barata et al. / Cancer Treatment Reviews 50 (2016) 35–47

Fig. 1. Biogenesis of different RNA molecules and pathways. 1. siRNA biogenesis and pathway. After being transcribed, double-stranded RNAs (dsRNAs) or short-hairpin RNAs (shRNA) exit the nucleus and are processed by a complex consisting of the endonuclease DICER into mature siRNA duplexes [6]. One of the double-stranded siRNA is then loaded into RISC complex, which contains the AGO family as a core component. The siRNA guide strand recognizes and links the target site by base pairing, to direct mRNA cleavage and degradation [10]. The catalytic domain of AGO2 carries out this degradation. 2. miRNA biogenesis and pathway. Primary miRNA transcripts (pri-miRNA) are originally transcribed from introns. Pri-miRNA are then cleaved by DROSHA and other partner proteins (i.e. DGC-8) into a loop precursor miRNA (pre-miRNA). After being exported to cytosol, DICER processes it and a mature miRNA is formed. One of the double-stranded miRNA is loaded into RISC complex. In the cytoplasm, the complex predominantly bind to 3´-UTR (untranslated region) of target mRNA (1) and repress its expression through translational repression and mRNA destabilization mechanism. Some miRNA have the ability to bind to the ORF (2) and the 5´-UTR (3) of the target mRNA, and activate or repress its translational efficiency. In the nucleus, miRNA bind to gene promoter to regulate gene expression (4). Lastly, miRNA can also be involved in inter-cellular communication after being secreted in the extracellular space (5) [166]. 3. AONs pathway. From the extracellular space, AONs interact with the cell surface and use different mechanisms (natural processes or facilitators) to enter the cell. When in the cytoplasm, can bind the target mRNA. The formation of AON-mRNA duplex activates RNase H enzyme, which cuts the mRNA preventing the synthesis of the protein. AON can also dissociate from mRNA and recycle [8,15].

for abstracts from the meetings of the American Society of Clinical Oncology, American Society of Hematology, American Association for Cancer Research, and European Society for Clinical Oncology, all from their respective inception to February 2016. The search terms were ‘‘medicine,” ‘‘cancer,” ‘‘clinical trial,” ‘‘phase 1” (or I), ‘‘phase 2” (II), ‘‘phase 3” (III), ‘‘first-in-human,” ‘‘RNA,” ‘‘RNAi,” ‘‘siRNA,” ‘‘microRNA,” and ‘‘antisense oligonucleotides.” All clinical trials on solid tumors and hematologic malignancies with published data were considered, including those with active and terminated status. Clinical trials of RNA-based vaccines or those focused on DNA targets, target cells ex vivo, ribozymes, or nonclinical measures (e.g., biomarkers) were excluded from this review. Current clinical studies of either RNAi or ASOs for the treatment of cancer are summarized in Tables 1 and 2. Our literature search uncovered 33 drugs, divided into 3 groups: siRNA/ dicersubstrate siRNA (dsiRNA) drugs, miRNA drugs, and mRNA ASOs. siRNA/dsiRNA drugs siRNAs are a class of double-stranded synthetic RNA molecules that can interfere with the expression of specific genes through RNAi (Fig. 1) [17]. siRNA libraries have been created to dissect

the function of independent genes, including targeting genes within animal and human cells, in conjunction with several cellbased assays [18]. The application of these platforms allows researchers to discover novel targets and pathway mediators [19,20]. Although this approach is promising, several challenges have been identified, including lack of stability against extracellular and intracellular degradation by nucleases, poor uptake and low potency at target sites of siRNAs, and off-target effects [21]. The pursuit of clinically viable antisense drugs has led to the development of various strategies to overcome these problems. Various carriers of siRNA have increasingly become available because RNAi can integrate short hairpin RNA into the cell genome, leading to stable siRNA expression and long-term knockdown of a target gene [1,11]. These carriers typically involve a positively charged vector (e.g., cationic cell-penetrating peptides, cationic polymers and lipids), small molecules (e.g., cholesterol, bile acids, lipids, and PEGylated lipids), polymers, antibodies, and lipid and polymerbased nanocarriers encapsulating the siRNA [1,22]. Despite the challenges, the effectiveness of siRNA therapies has been extensively demonstrated in preclinical studies, and some of the agents have already entered clinical trials for the treatment of

CR: complete response; GGT: gamma-glutamyl transferase; ICE: inflammatory cytokine elevation; LEE: liver enzymes elevated; LODER: local drug eluter; n.s.: not specified; PKN3: protein kinase N3; PLK1: polo-like kinase 1; PR: partial response; RRM2: ribonucleotide reductase M2 subunit; SD: stable disease.

Beg et al. [138] Van Zandwijk et al. [139] Neutrophilia, lymphopenia, hyponatremia, LEE, ICE Neutrophilia, lymphopenia, LEE, ICE 60% quality of life improvement 3% PR I I Lipid nanoparticles Drug-loaded nanoparticles miR-34 miR-16 miRNA replacement MRX34 TargomiRs

Advanced cancers Lung/pleura

RRM2 CALAA-1

Cyclodextrin nanoparticles

I

No objective responses

Schultheis et al. [29] Cervantes et al. [26]

<10% G3 (n.s.), 2 G4 GGT and lipase elevation 1 death, 2 DLT = 1 G3 thrombocytopenia, 1 G3 hypokalemia, 10% ICE Closed owing to DLTs: 50% fatigue, 42% fever Lipid nanoparticles Lipid nanoparticles PKN3 VEGF, KSP Solid tumors Atu027 ALN-VSP02

siGD12 LODER Atu027

I I

Golan et al. [28] Schultheis et al. [30] Transient G1/2 abdominal pain, diarrhea, nausea 17% G4 and82–92% G3 events (n.s.)

17% PR, 83% SD Progression-free survival not statistically different 41% SD 8% PR, 47% SD I/II I/II LODER polymer Lipid nanoparticles KRAS PKN3

Tolcher et al. [32] Rolle et al. [31] Ramesh et al. [25] 1 DLT = G3 LEE; G1-2 fatigue, ICE No significant neurotoxicity Transient G1/2 nausea/vomiting, fatigue, ICE 1 CR, multiple PR (n.s.) Better overall survival, quality of life 3% PR, 11% SD I/II I I/II Lipid nanoparticles Naked molecule Lipid nanoparticles Myc Tenascin-C PLK1

Advanced cancers Brain Neuroendocrine/ adrenal Pancreas siRNA/dsiRNA DCR-MYC ATN-RNA TKM 080301

Citations Most common or dose-limiting toxicity (DLT) Best response Phase of study Delivery platform Target Tumor type Drug

Table 1 MicroRNA (miRNA) and short interfering RNA (siRNA)/dicer-substrate siRNA (dsiRNA) therapeutics in clinical trials.

Zuckerman et al. [27]

P. Barata et al. / Cancer Treatment Reviews 50 (2016) 35–47

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cancer and several other diseases [23,24]. To date, 8 clinical trials of siRNA/dsiRNA therapies for cancer have been published (Table 1). Various siRNA targets or pathways were considered, including (1) cell-cycle: polo-like kinase 1 (PLK1) [25], kinesin spindle protein (KSP) [26], and ribonucleotide reductase M2 subunit (RRM2) [27]; (2) signaling: KRAS(G12D) [28]; (3) proliferation: protein kinase N3 (PKN3) [29,30] and tenascin C [31]; and (4) angiogenesis: vascular endothelial growth factor (VEGF) [26]. In addition, preliminary results from a phase I trial with the first dsiRNA-based therapy that targets the oncogene MYC, in patients with advanced solid tumors, have been published (NCT02110563) [32]. These data must be interpreted with caution because they are early-phase trials and many are still recruiting patients. The best responses observed so far have been tumor stabilization, with very few complete or partial responses documented. One exception was local administration of the dsiRNA ATN-RNA after brain surgery, which led to a significant improvement in overall survival and better quality of life [31]. The other trial of a dsiRNA, DCR-MYC, reported a number of partial responses and one complete response to treatment in patients with advanced cancer [32]. siRNAs were well tolerated; adverse effects mostly included mild fatigue and elevation of inflammatory cytokines. However, one death and a few grade 3–4 toxic effects due to elevation of liver enzymes were also observed [26,29,32]. In most of these studies, pharmacokinetics data, including the area under the curve and maximal concentration of these molecules, were studied with pretreatment and posttreatment biopsies as part of contemporary phase I safety trials. In a few cases, plasma siRNA samples were used, such as the studies of CALAA-1. Downregulation of the target of interest was reported only in 2 drugs: Atu027, for which soluble variant of vascular endothelial growth factor receptor-1 (sFLT1) was measured, and ATN-RNA, for which tenascin C expression levels were measured and controlled.

miRNA drugs miRNAs are small noncoding RNAs that regulate a variety of biological processes, including apoptosis, survival, senescence, and metabolism [33,34]. Because miRNAs are frequently located at fragile sites of the human genome, they may be aberrantly expressed in tumors [35–38]. The fact that miRNA deregulation is very common in human disease has led to increasing interest in therapeutic targeting of miRNAs, as indicated by the number of PubMed hits (
45,990) we found for peer-reviewed scientific literature on miRNA (as of February 2016). Numerous preclinical studies in cells and animal models have been conducted, and miRNA was shown to be an effective therapeutic target [6,18,39]. Drugs targeting miRNAs then moved to clinical research, and two trials using miRNA mimics in patients with cancer have been reported (Table 1). The targets were miR-16, a tumor suppressor of cell proliferation and tumorigenicity [40], and miR34, a key controller of several proteins involved in the cell cycle [41]. It seems too early to evaluate the efficacy of these drugs, given that the trials are ongoing and data are preliminary; however, in both cases, the therapies were well tolerated, with minor transient inflammatory reactions reported. Several other trials are ongoing, both in cancer and non-cancer populations [2]. Because miRNAs have the unique advantage of targeting multiple genes, various options are being explored to improve the clinical efficacy of these drugs in cancer [42]. Current advances include the combination of miRNA with other anticancer therapies such as siRNAs and exploring potential synergistic effects on reducing tumor growth, such as miR-520d combined with EPH2 [43]. The design of novel delivery systems to overcome the vulnerability of

Tumor type Advanced cancers Solid tumors

Drug

Target

Phase of study

Best response

Most common or dose-limiting toxicity (DLT)

Citations

LY2181308 LErafAON-ETU ISIS 5132 (every 3 weeks)

survivin c-raf-1 c-raf-1

I I I

8% SD No objective responses 6% SD

G3 14% LEE; G1/2 fatigue, TP, flu-like syndrome Reversible IRR G1/2 fever, fatigue

ISIS 5132 (weekly) ISIS 5132 + carboplatin + paclitaxel Aprinocarsen + 5-fluorouracil ISIS 183750 + irinotecan

c-raf-1 c-raf-1 PKC-a elF4E

I I I I/II

No objective responses No objective responses 13% PR, 27% SD No objective responses

DLT 9% ARI, anasarca, hemolytic anemia 2 DLT: neutropenia G4/3 chest pain, mucositis, neutropenia G3/4 33% neutropenia, 25% hypoalbuminemia

Tanioka et al. [109] Rudin et al. [140] Stevenson et al. [141] Rudin et al. [142] Fidias et al. [143] Mani et al. [144] Makarova-Rusher et al. [145]

GTI-2040 GTI-2040 + docetaxel GTI-2040 GTI-2040 + capecitabine + oxaliplatin ISIS 2503 + gemcitabine Oblimersen Oblimersen + carboplatin + paclitaxel Oblimersen + gemcitabine Oblimersen + doxorubicin + cyclophosphamide Apatorsen + docetaxel Custirsen + docetaxel

RRM2 RRM2 RRM2 RRM2 H-ras bcl-2 bcl-2 bcl-2 bcl-2

I I/II I I I I I I I

No objective responses No objective responses 17% PR, 33% SD 4% PR, 19% SD No objective responses 13% No objective responses 73% biologic response

1 death: FN; G1/2 fatigue, alopecia, leukopenia 2 DLT: LEE DLT: 20% TP G3/4 neutropenia, TP 66% fatigue, 40% LEE, 46% anemia 2 DLT: neutropenia, TP 1 DLT: edema; fatigue, N/V, myalgias 2 DLT: neutropenia, TP

Hsp27 clusterin

I I

23% PR, 13% SD 6% PR, 34% SD

Trabedersen GEM231 GEM231 + docetaxel AEG35156 AEG35156 + docetaxel RX-0201

TGF-b2 PKA-I PKA-I XIAP XIAP Akt-1

I/II I I I I I

3% CR, median OS > 1 year No objective responses No objective responses 3% PR 5% PR, 18% SD No objective responses

1 DLT: cerebral hemorrhage Myelosuppression, fatigue, gastrointestinal-related symptoms, hair loss G1/2 TP G1/2 fatigue, APTT elevation 3 DLT: neutropenia, LEE, fatigue 3 DLT: lethargy, LEE; G1 chills, APTT elevation, HEE DLT: LEE, TP, hypophosphatemia G1 fatigue, dizziness, cough, myalgia

LErafAON-ETU + radiation therapy

c-raf-1

I

33% PR, 33% SD

G3/4 IRR; G1/2 chills, dyspnea, hypertension, back pain

AZD9150

STAT3

I

1 DLT: TMA; G3 TP

LY2275796 Aprinocarsen ISIS 5132

elF-4E PKC-a c-raf-1

I I I

66% PR in DLBCL; no objective responses in solid tumors 23% SD No objective responses 3% PR, 7% SD

Imetelstat

telomerase

I

10% PR

33% DLT: myelosuppression

Veglin

I

2% CR, 2% PR

II

No objective responses

1 DLT: fever + PE; G1/2 anemia, fatigue, gastrointestinalrelated symptoms, fever G3/4 LEE, TP

G3 TP, lymphopenia; G1/2 fatigue, N/V, fever 1 G4: neutropenia; G3 fever, hemorrhage, N/V No significant toxicities

Leighl et al. [146] Desai et al. [147] Shibata et al. [148] Adjei et al. [149] Morris et al. [150] Liu et al. [151] Galatin et al. [152] Rheingold et al. [153] Hotte et al. [154] Chi et al. [155] Oettle et al. [95] Chen et al. [156] Goel et al. [157] Jolivet et al. [158] Dean et al. [159] Malik et al. [160] Dritschilo et al. [137] Hong et al. [121] Hong et al. [58] Advani et al. [161] Cunningham et al. [162] Thompson et al. [163] Levine et al. [98]

Brain

Aprinocarsen

VEGF-A, -B, -C, -D PKC-a

High-grade glioma

Trabedersen vs chemotherapy Trabedersen vs chemotherapy

TGF-b2 TGF-b2

I/II II

4% CR, 8% > 80% PR No difference in control rate at 6 months

4% G3 G3/4 76%: mostly nervous system-related

ISIS 2503

H-ras

II

No objective responses

No significant toxicities

Aprinocarsen vs ISIS 5132

PKC-a vscraf-1 bcl-2 bcl-2

II

No objective responses

1 DLT: TP; G1/2 fatigue, anorexia, N/V

Marshall et al. [164] Cripps et al. [55]

I/II I/II

5% PR, 50% SD 7% CR, 13% PR

G3/4 FN, diarrhea; G1/2 fatigue, fever, N/V G3/4 neutropenia, hypokalemia, infection, mucositis

Mita et al. [53] Raab et al. [52]

Gastrointestinal cancers Colorectal

Esophageal-gastric junction

Oblimersen + irinotecan Oblimersen + cisplatin + fluorouracil

Grossman et al. [91] Bogdahn et al. [93] Bogdahn et al. [94]

P. Barata et al. / Cancer Treatment Reviews 50 (2016) 35–47

Solid and liquid tumors

38

Table 2 mRNA antisense oligonucleotide therapeutics in clinical trials, for various cancer sites.

Merkel cell

Oblimersen

bcl-2

II

No objective responses

Pancreas

GEM640

XIAP

I/II

36% SD

G3/4 myelosuppression, renal failure, LEE, hypophosphatemia G3/4 TP, peripheral, neuropathy, N/V, fatigue, ascites

ISIS 2503 + gemcitabine

Shah et al. [54]

H-ras

II

2% CR, 8% PR

1% G3/4 PE

Mahadevan et al. [51] Alberts et al. [56]

Gynecologic and genitourinary malignancies Women Breast Oblimersen + chemotherapy Custirsen + chemotherapy Imetelstat ± chemotherapy Imetelstat ± trastuzumab Ovarian Aprinocarsen

bcl-2 clusterin telomerase telomerase c-raf-1

I/II II I II II

No objective responses 33% response rate 7% CR, 29% PR, 36% SD No objective responses No objective responses

G3/4 hematologic malignancies Similar toxicities in two arms G2 neutropenia/TP; G P 2 fatigue, N/V, diarrhea G2/3 anemia, TP 21% G3 fatigue, abdominal pain, anorexia, dyspnea

Moulder et al. [64] Chia et al. [67] Kozloff et al. [71] Miller et al. [70] Oza et al. [165]

Men Kidney

GTI-2040 ± CT

RRM2

I/II

4% RR

Stadler et al. [76]

RX-0201 + everolimus Oblimersen + interferon Apatorsen + prednisone Oblimersen + docetaxel

Akt-1 bcl-2 Hsp27 bcl-2

I/II II II I

Oblimersen + docetaxel

bcl-2

II

40% SD 4% PR 38% PR 58% PSA responses in chemotherapynaive <30% PSA responses

Phase I: 17% G3/4 diarrhea; phase II: 9% sepsis; G1/2 nausea, fatigue, constipation, anemia TP, fatigue, vomiting; no DLT G3/4 fever, LEE, fatigue, myelosuppression 47% G1/2 IRR G1/2 fever, N/V 41% G3/4 fatigue, mucositis, TP

Oblimersen + mitoxantrone EZN-4176 Custirsen + androgen antagonist Custirsen + docetaxel/mitoxantrone Custirsen + docetaxel

bcl-2 AR clusterin clusterin clusterin

I I I II III

Aprinocarsen vs ISIS 5132

PKC-a vs craf-1 survivin RRM2 Hsp27

Prostate

G1/2 fatigue, arthralgias, nausea, myalgias; no DLT G1/2 IRR, fatigue, LEE; 2 DLT: LEE G1/2; no DLT Similar toxicities in two arms 13% increase in G3/4 myelosuppression

II

8% PSA response>50% No objective responses >90% clusterin response 23% PR, 60% PSA response > 50% OS HR 0.72, PFS HR 0.73 prognosispoor prostate cancer No PSA responses

Sternberg et al. [62] Chi et al. [65] Bianchini et al. [75] Chi et al. [68] Saad et al. [66] Chi et al. [69]

G1/2 fatigue, lethargy

Tolcher et al. [73]

II II II

No difference in outcomes 6% PR, 75% SD,41% PSA response No difference in PFS/OS

G3/4 myelosuppression, sensory neuropathy G3/4 myelosuppression, fatigue, hypophosphatemia G3 myelosuppression, HBP

Wiechno et al. [72] Sridhar et al. [77] Bellmunt et al. [60]

grb-2

I

29% PR/SD

1 DLT (mucositis, HFS)

P53 P53 RRM2

I II I

Suppression of leukemic cell growth 15% CR, 4% CRi 35% CR

No significant toxicities No significant toxicities DLT: neurotoxicity

Ohanian et al. [106] Bishop et al. [108] Cortes et al. [105] Klisovic et al. [166]

RRM2

I

No CR

1 toxic death (hemorrhage), 1 G4 sepsis, 31% fatigue, FN

Klisovic et al. [107]

RRM2 bcl-2

II I

26% CR/CRi, 4% PR 41% CR

Similar toxicities in two arms Similar to chemotherapy alone

Oblimersen + gemtuzumab ozogamicin, >60 years Oblimersen + chemotherapy,>60 years

bcl-2

II

25% CR/CRi

Similar to chemotherapy alone

Klisovic et al. [111] Marcucci et al. [100] Moore et al. [112]

bcl-2

III

No difference in outcomes

Similar toxicities in two arms

LY2181308 + idarubicin + cytarabine AEG35156 + idarubicin + cytarabine

survivin XIAP

I I/II

25% CR, 6% PR High-dose group: 47% CR/CRi

G3/4 33% FN, 21% sepsis G3/4 8% neutropenia

AEG35156 + idarubicin + cytarabine

XIAP

II

No difference in remission rates

EZN-3042 + reinduction chemotherapy Oblimersen + imatinib

survivin bcl-2

I I

No objective response 10% responses

3 toxic deaths: 2 sepsis,1 gastrointestinal hemorrhage; G3/4 33% diarrhea, anorexia 33% DLTs: LEE, 1 gastrointestinal hemorrhage G3/4 19% fatigue, 38% neutropenia, 24% TP

LY2181308 GTI-2040 + docetaxel Apatorsen + gemcitabine + cisplatin

Hematologic malignancies Leukemia (AML/ALL/ BP-100–1.01 CML) + MDS AML + MDS Cenersen AML Cenersen + idarubicin + cytarabine GTI-2040 + high-dose cytarabine, <60 years GTI-2040 + high-dose cytarabine > 60 years GTI-2040 + high-dose cytarabine Oblimersen + paclitaxel

ALL CML

P. Barata et al. / Cancer Treatment Reviews 50 (2016) 35–47

Urothelial

Tagawa et al. [74] Margolin et al. [63] Chi et al. [59] Tolcher et al. [61]

Marcucci et al. [113] Erba et al. [103] Schimmer et al. [104] Schimmer et al. [110] Raetz et al. [167] Wetzler et al. [115] (continued on next page) 39

40

Table 2 (continued) Drug

Target

Phase of study

Best response

Most common or dose-limiting toxicity (DLT)

Citations

CLL

Oblimersen Oblimersen + cyclophosphamide + fludarabine SPC2996 Cenersen +cyclophosphamide + fludarabine + rituximab Oblimersen

bcl-2 bcl-2

I/II III

DLT: hypotension, fever TP

O’Brien et al. [168] O’Brien et al. [117]

bcl-2 P53

I/II II

8% PR Better OS (HR 0.6) for patients with CR/PR 100% lymphocyte reduction count 53% response rate, 18% CR

2 DLTs in higher escalation group G3/4 TP, neutropenia

Tilly et al. [116] Lanasa et al. [169]

bcl-2

I

5% CR, 10% PR, 43% SD

DLT: TP, hypotension, fever; 100% local skin inflammation

Waters et al. [118]

Oblimersen ± R-CHOP

bcl-2

I/II

Similar toxicities in two arms

Leonard et al. [170]

Oblimersen + rituximab Aprinocarsen Imetelstat + bortezomib

bcl-2 PKC-a Telomerase

II II I

75% response rate in combination arm 21% CR, 17% PR, 25% SD 13% PR, 30% SD n.s.

Reversible G1/2 G3/4 4% neutropenia, 27% TP G3/4 TP, neutropenia, anorexia, APTT prolongation

Oblimersen + thalidomide + dexamethasone Oblimersen + dexamethasone

bcl-2

II

18% CR/CRi, 36% PR

G3/4 neutropenia, TP, hypocalcemia, infection

Pro et al. [120] Rao et al. [102] Chanan-Khan et al. [101] Badros et al. [171]

bcl-2

III

No difference in outcomes

Fatigue, fever, nausea

Chanan-Khan et al. [114]

Oblimersen + temozolomide + nabpaclitaxel Oblimersen + dacarbazine Oblimersen + dacarbazine

bcl-2

I

6% CR, 34% PR, 34% SD

6% G4 neutropenia, TP; G3 neuropathy, hyponatremia, ARI

Ott et al. [79]

bcl-2 bcl-2

I/II III

G1/2 LEE, lymphopenia; no DLTs Same G3/4; G1/2 neutropenia, TP

Jansen et al. [81] Bedikian et al. [80]

Oblimersen + dacarbazine (normal/low lactate dehydrogenase)

bcl-2

III

7% CR, 14% PR No difference in OS, better PFS/ overall response rate No difference in clinical outcomes

TP, neutropenia more frequent

Bedikian et al. [99]

ISIS 5132 + CT Oblimersen + paclitaxel Oblimersen + carboplatin + etoposide Custirsen + gemcitabine + cisplatin LY2181308 + docetaxel Aprinocarsen + gemcitabine + cisplatin

c-raf-1 bcl-2 bcl-2 clusterin survivin PKC-a

II I II I/II II I/II

No objective responses No objective responses No difference in outcomes 31% response rate No difference in outcomes 3% CR, 33% PR

4% elevated PT; G1/2 fatigue, N/V G3/4 8% PE, 17% myelosuppression,58% G1/2 fatigue 28% increase in G3/4 myelosuppression No significant toxicities Similar G3/4 in both arms G3/4 57% neutropenia, 71% TP

Aprinocarsen + gemcitabine + cisplatin

PKC-a

II

No difference in outcomes

52% increase in G3/4 TP

Aprinocarsen + gemcitabine + cisplatin Aprinocarsen + gemcitabine + carboplatin Aprinocarsen + carboplatin + paclitaxel Aprinocarsen + carboplatin + paclitaxel Imetelstat + bevacizumab

PKC-a PKC-a PKC-a PKC-a Telomerase

III II I/II III II

No difference in outcomes 25% PR, 36% SD 46% response rate No difference in outcomes No difference in outcomes

30% increase in G3/4 TP,G3/4 1% epistaxis G3/4 78% TP, 50% neutropenia G3/4 21% TP, 26% neutropenia n.s. G3/4 35% TP, 17% neutropenia

Coudert et al. [92] Rudin et al. [97] Rudin et al. [78] Laskin et al. [82] Natale et al. [84] Villalona-Calero et al. [87] Vansteenkiste et al. [88] Paz-Ares et al. [86] Ritch et al. [85] Yuen et al. [89] Lynch et al. [96] Chiappori et al. [83]

Non-Hodgkin lymphoma Mantle-cell leukemia

Low-grade nonHodgkin lymphoma Multiple myeloma

Melanoma

Lung All SCLC NSCLC

AA: androgen antagonist; AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia; APTT: activated partial thromboplastin time; AR: androgen receptor; ARI: acute renal impairment; CML: chronic myeloid leukemia; CLL: chronic lymphoblastic leukemia; CR: complete response; CRi: incomplete response; CT: chemotherapy; DLBCL: diffuse large B cell lymphoma; FN: febrile neutropenia; HFS: hand-foot syndrome; HBP: high blood pressure; HR: hazard ratio; IRR: infusion-related reactions (chills, flushing, pyrexia, nausea, diarrhea); LEE: liver enzymes elevated; MDS: myelodysplastic syndrome; N/V: nausea/vomiting; n.s.: not specified; NSCLC: non-small cell lung carcinoma; OS: overall survival; PE: pulmonary embolism; PFS: progression-free survival; PKA-I: cAMP dependent protein kinase type I; PKC-a: protein kinase C alpha; PR: partial response; PSA: prostate-specific antigen; PT: prothrombin time; RRM2: ribonucleotide reductase M2 subunit; R-CHOP: rituximab + cyclophosphamide + doxorubicin + vincristine + prednisone; SCLC: small-cell lung carcinoma; SD, stable disease; TMA: thrombotic microangiopathy; TP: thrombocytopenia; VEGF: vascular endothelial growth factor; XIAP: X-linked inhibitor of apoptosis protein.

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Tumor type

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RNA molecules has also improved with the use of nanoparticle platforms mentioned above, and the 2 drugs studied in cancer trials (Table 1) both used these platforms.

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in patients with colorectal cancers or other advanced malignancies is ongoing (NCT01675128). Genitourinary and gynecologic cancers

mRNA ASOs Among all RNA-based therapies, ASOs are the group of drugs with the longest track record, having undergone investigation for more than 20 years [44]. Over time, the pursuit of effective antisense drugs has led to modifications in their structure. The first of these modifications included phosphorothioate backbone modification, which defined the first-generation ASOs [44,45]. Because many drugs failed to achieve the desired effect without major toxicities, it became clear that a number of obstacles needed to be adressed [46]. Some advances included the synthesis of nucleoside analogues containing a modified sugar moiety, such as 20 -O-methyl-modified (20 -OMe) or 20 -O-methoxyethyl (20 -OMOE) [9,47–49]. These second-generation ASOs addressed the problems of low cellular uptake, lack of specificity, and poor affinity to target RNA. However, all 20 modifications inhibit the ability of RNase H to cleave the bound ‘‘sense” RNA strand within the heteroduplex formed between the ASO and the target RNA [46]. This limitation was somewhat addressed with the use of the chimeric gapmer strategy, in which the 20 regions of a synthetic ASO flank a central DNA region called the ‘‘gap.” Finally, to increase thermal stability when ASOs were hybridized to complementary RNAs, researchers developed several nucleic acid analogs, including peptide nucleic acids and locked nucleic acids [50]. ASOs have been tested in more than 100 clinical trials. Here, we briefly summarize the data available according to tumor type. The information in Table 2 is organized the same way, to facilitate reading. Gastrointestinal malignancies Five different ASOs have reached clinical trials for the treatment of gastrointestinal malignancies (Table 2). The targets of these molecules were related to cell proliferation—X-linked inhibitor of apoptosis protein (XIAP) [51], bcl-2 [52–54], and protein kinase C alpha (PKC-a) [55]—or cell signaling—H-ras [56] and c-raf-1 [55]. Among all ASOs tested, only ISIS 2503 showed some evidence of growth inhibition, when combined with gemcitabine in locally advanced or metastatic pancreatic cancer in first-line treatment. In that study, 58% of patients who received the combination survived 6 months or longer, representing an increase of about 12% in overall survival rates compared with historic gemcitabine pivotal trials [57]. On the basis of these results, various strategies were considered for additional testing of ISIS 2503. However, no phase III trial was started, possibly because other novel treatment options were approved for pancreatic cancer since then. In a trial of oblimersen, no objective responses were detected in colorectal or Merkel cell cancers. In patients with cancer of the gastroesophageal junction, although objective responses were observed in 20% of cases, the sample size was insufficient to determine the efficacy of the combination of oblimersen with chemotherapy. Moreover, the phase II part of the study was discontinued because of uncertainty about the future development of oblimersen after a phase III trial of this molecule in melanoma showed negative results, as noted below [52]. The second-generation ASO LY2275796 was first tested in humans with advanced solid tumors, including colon cancer [58]. In that trial, no objective responses were seen, but tumor elF-4E expression was decreased, suggesting a possible antitumor effect when LY2275796 was combined with other treatment modalities. Currently, a trial of the combination of LY2275796 with irinotecan

Published data indicate that 9 ASOs, alone or combined with other agents, have been tested in the setting of genitourinary and gynecologic cancers. The targets of these drugs are related to cell proliferation-heat shock protein 27 (Hsp27) [59,60], bcl-2 [61–65], clusterin [66–69], telomerase [70,71], survivin [72], and protein kinase C alpha (PKC-a) [73]; cell signaling—Akt-1 [74] and AR [75]; or the cell cycle—RRM2 [76,77] (Table 2). Prostate cancer is the genitourinary tumor most frequently targeted by ASOs in published studies. To date, custirsen was the most successful agent, with favorable toxicity profiles and positive results in phase I/II trials [66,68]. The modulation effect of this drug was studied by measuring clusterin expression in both the tumor tissue and serum of patients with prostate cancer. In that study, an on-treatment dose-dependent and exposure-dependent decrease in tumor burden was observed, but the correlation between serum levels of clusterin and levels in the tumor tissue was not established. Despite the positive results in both progression-free and overall survival in patients with poor prognosis, treatment with custirsen in this phase III trial failed to extend survival in the first-line setting, when combined with docetaxel [69]. A phase III trial in the second-line setting is ongoing (NCT01578655). Oblimersen has also been tested in prostate cancer, but the primary endpoints of the phase II study [62] were not met. Despite interesting results in phase I studies showing that bcl-2 protein expression in tumor specimens decreased, this decrease was not predictive of response to therapy [61,65]. Apatorsen was tested in three phase II trials, one for prostate cancer and two for bladder cancer. Although preliminary data from the prostate cancer trial seem to indicate tumor stabilization with a favorable toxicity profile [59], results from the bladder cancer trials showed no differences in outcomes compared with standard gemcitabine plus cisplatin [60]. Another phase II trial comparing apatorsen with docetaxel in patients with relapsed or refractory bladder cancer after receiving a platinum-containing regimen is ongoing (NCT01780545). In kidney cancer, the combination of RX-0201 with everolimus in the second-line setting or beyond appears to be well tolerated; however, no information on efficacy is available because the trial in the dose escalation cohort is still ongoing. Finally, in breast and ovarian cancers, all trials have failed to show improved clinical outcomes and no further development is planned at this time. Here, the targets of these drugs showed very little downregulation or, when present, were not correlated with response to these therapies. Brain, lung, and skin malignancies Five of the seven drugs tested for brain, lung, or skin malignancies target cell proliferation—bcl-2 [78–81], clusterin [82], telomerase [83], survivin [84], and protein kinase C alpha (PKC-a) [85–91]—and the remaining two target cell signaling—ISIS 5132, which targets c-raf-1 [92], and trabedersen, which targets TGF-b2 [93,94]. As seen in other tumors, intratumoral administration of trabedersen led to shrinkage of the target tumor as well as tumors elsewhere in the brain in phase I/II and phase II trials [93,95]. The long-term benefit detected in the randomized phase II trial prompted the researchers to study trabedersen in a phase III trial, comparing trabedersen with temozolomide (NCT00761280) in the first-line setting [94]. However, mainly owing to other advances in

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both surgical procedures and first-line standard of care for patients with glioblastoma multiforme, the trial was halted owing to patient recruitment issues. In non-small cell lung cancer, the first-generation ASO aprinocarsen has been the therapy of choice, alone or in combination with various chemotherapy agents. In early-phase studies, aprinocarsen showed activity when used in combination with chemotherapy. However, it proved to be disappointing in two phase III trials, with no improvement in outcomes and higher toxicity when combined with two parallel chemotherapy regimens [86,96]. In small-cell lung cancer, oblimersen showed no tumor activity, either alone or in combination with various chemotherapy regimens [78,97]. In mesothelioma, modest responses were also documented with veglin, an angiogenesis inhibitor, in a phase I trial including patients with various types of tumors [98]. Combinations of veglin with various chemotherapy agents, depending on the tumor type, were designed, but no results have been published yet. In melanoma, results of a phase I/II study with oblimersen showed that reducing bcl-2 concomitant to the administration of dacarbazine could amplify apoptosis and improve clinical outcomes [81]. In that study, a median 40% decrease in bcl-2 protein in melanoma samples compared with baseline was observed, as well as tumor-cell apoptosis. These findings led to further study of this molecule. In 2000, the first phase III study started, comparing oblimersen plus dacarbazine with dacarbazine alone in patients with advanced melanoma. Progression-free survival time was statistically longer but not median overall survival time, although a correlation between pretreatment lactate dehydrogenase level and outcome was detected. Therefore, a second phase III trial studying the addition of oblimersen to dacarbazine in patients with melanoma and low baseline lactate dehydrogenase was conducted [99]. This trial also showed negative results; progression-free and overall survival did not differ between treatments and increased toxicity with the combination was observed. In the past few years, the standard of care in melanoma has changed and new drugs have been approved. For this reason, the combination of oblimersen with temozolomide and nabpaclitaxel was recently tested, and this combination showed encouraging activity, with 6% showing complete responses and 50% of patients surviving >1 year [79]. Further development of this and other combinations is currently being explored. Hematologic malignancies Several ASOs were tested in liquid tumors, including those with cell proliferation targets—bcl-2 [100], telomerase [101], PKC-a [102], survivin [103], XIAP [104], p53 [105], and Grb-2 [106]—and those with cell-cycle targets—GTI-2040 (RRM2) [107]. In acute myeloid leukemia (AML), both cenersen and LY2181308 showed potential antileukemic effect in phase I trials [108,109] and were tested in combination with chemotherapy in different phase II trials [105]. Although cenersen showed some evidence of clinical activity, results from the study with LY2181308 are not known (NCT00620321). Although AEG35156 was effective in inducing apoptosis of AML stem cells, with evidence of XIAP mRNA knockdown at higher doses, the development of this drug was discontinued after excessive severe toxicity and no improvements in remission rates were reported [104,110]. The ASO GTI-2040 was also studied in refractory or relapsed AML, in combination with backbone high-dose cytarabine. After this combination failed to show clinical responses in the first study in elderly patients [107], the same combination was subsequently studied on a different schedule; clinical efficacy was evident in this setting, with no major toxicities [111], In these studies, bone marrow lysates were collected before and after treatment, and

evidence of intracellular drug accumulation and target R2 downregulation were observed. Finally, oblimersen was also (extensively) tested in patients with AML. Indeed, an antitumor effect in AML was found when oblimersen was combined with chemotherapy, and a significant number of complete responses occurred in elderly patients, even though plasma pharmacokinetics did not correlate with disease response and changes in bcl-2 protein showed a similar trend [100,112]. To confirm these results, a phase III trial was conducted in patients older than 60 years with AML, but there was no change in clinical outcomes compared with chemotherapy alone [113]. Likewise, after oblimersen was shown to have some efficacy in patients with refractory multiple myeloma, the combination of oblimersen with dexamethasone did not change time to tumor progression in another phase III trial [114]. This same drug was also tested in patients with chronic myeloid leukemia (CML) in combination with imatinib, but again, very few responses were observed [115]. In contrast, both preclinical data and results from early trials in patients with refractory chronic lymphocytic leukemia (CLL) suggested that oblimersen might enhance the apoptotic response in CLL cells to chemotherapy [116]. The major responses observed led to the initiation of a phase III trial exploring the addition of oblimersen to fludarabine and cyclophosphamide in patients with relapsed or refractory CLL [117]. In that trial, the 5-year overall survival analysis showed no significant difference between treatment arms; however, in the subset of patients with partial or complete remission, a significant 5-year overall survival benefit was noted. On the basis of these results, a request for US Food and Drug Administration (FDA) approval was made in 2006. However, 3 years later, the FDA final decision indicated that a confirmatory clinical trial was recommended. The ASO BP-100-1.01 has also been studied in patients with leukemia, including AML, CML, and ALL, and data from the phase I study were recently presented [106]. The drug has been very well tolerated, with minimal dose-limiting toxicities, and grb-2 target protein downregulation was linked with anti-leukemic activity in about one-third of the patients. These results support further studies of this molecule, as a phase Ib/II trial (NCT 01159028) with BP1001 in combination with low dose cytarabine is ongoing. In non-Hodgkin lymphomas, preclinical data support the chemosensitizing potential of oblimersen, which was shown to enhance the cytotoxicity of a variety of other agents, in contrast with its modest activity as a single agent [118,119]. The reduction of bcl-2 protein observed in this setting suggested a specific antisense mechanism, and additional studies will determine whether the addition of this antiapoptotic agent can increase the clinical efficacy of standard chemotherapy regimens [118,120]. The first-in-class telomerase inhibitor imetelstat has been tested in various high-risk myeloid malignancies [101]. The persistent low-grade hepatotoxicity observed in most patients enrolled led the FDA to place this drug on clinical hold for several years. After the FDA removed the clinical hold in 2014, the clinical trial of imetelstat in patients with myelodysplastic syndromes was reinitiated (NCT02598661), but no results are available. Finally, a first-in-class ASO targeting STAT3 was also studied in patients with various lymphomas, among other malignancies [121]. Although no objective responses were seen in solid tumors, a partial response in 66% of patients with diffuse large B cell lymphoma was observed, and a dose expansion trial in advanced lymphomas is ongoing. For reasons that are not completely understood, patients with hematologic malignancies appear to be more susceptible to adverse events (particularly sepsis and gastrointestinal hemorrhage) than those with solid tumors. Many, but not all, of these

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adverse events occurred with oblimersen, which was the most used ASO in various types of lymphomas and leukemias.

Lessons learned and future directions In the cancer world, less than two development drugs in ten (16%) that enter human clinical research reach phase III trials, and only about one in ten (10.4%) is expected to be approved by the FDA [122]. To the best of our knowledge, about 25% of the targets of the antisense drugs mentioned in this review were not explored in human clinical trials previously. Nevertheless, the success rate of these therapies reaching phase III trials is around 14%, and to date the FDA has approved none of these drugs. RNAi technology has the potential to play a major role in cancer. In fact, there are several aspects of antisense therapy that are potentially advantageous over traditional drugs. These include the ability to generate specific inhibitors of targets that were inaccessible until now, with the only limit being the genetic information available. Furthermore, inhibition of mRNA expression has the potential to produce faster and longer-lasting responses than protein inhibition by conventional targeted therapy. Moreover, antisense drugs have so far been well tolerated and safe, with few cases of transitory and mild toxicity documented. Lastly, oligonucleotides can be manufactured quickly and at lower costs than traditional small molecules; the sequence of the target mRNA is all that is needed and is relatively easy to obtain [123,124]. In addition to single gene targeting, RNAi is suited for coextinction or therapeutic synergy, which may represent an important step to overcome compensatory effects typically observed in cancer cells following knockdown of a single target [1]. Once a formulation proves to be efficacious and safe, the developmental path for subsequent targets is likely to be faster. Equally important, many of the approaches mentioned have focused on tumor cells as targets; precise approaches targeting other biological elements such as stromal cells are emerging [125]. The reasons for the low success rate with RNA therapeutics are not completely identified, but some important challenges have been addressed, including issues with delivery, targeting, and offtarget effects. Efficient and specific delivery is considered an important challenge for this class of drugs. Specific properties of biological organisms and cancer need to be addressed to optimize the cellular uptake of RNA drugs. In humans, ‘‘naked” ASOs, as well as various forms of miRNA, preferentially accumulate in the liver and kidneys, which causes the ASOs to be rapidly cleared from circulation, with poor tissue distribution [126]. In addition, the tumor environment is characterized by an enhanced permeability and retention effect, high interstitial fluid pressure, and asymmetrical leakiness of tumor vessels, which may all contribute to a highly heterogeneous process of drug penetration [127]. The pursuit of clinically viable antisense drugs has led to the development of various types of strategies, such as carriers or chemical modifications [9]. To promote metabolic stability and improve target cell penetration of siRNA, chemical modifications were developed, which led to different generations of ASOs, as explained above. The problem of instability has been addressed with various carriers of siRNA. Among these, nonviral vectors have been increasingly preferred owing to fewer toxicities compared with other carrier methods [128,129,22]. Various carriers are used at present, including those involving a positively charged vector (e.g., cationic cell-penetrating peptides, cationic polymers, and cationic lipids); small molecules (e.g., cholesterol, bile acids, lipids, and PEGylated lipids); polymers; antibodies; and nanoparticle formulations [11,130–132].

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Important modifications have been implemented to improve the therapeutic potential of antisense drugs. However, the properties of these modifications have also led to some decreased affinity for the target sequence, with associated non-hybridization toxicities, such as complement activation, increased coagulation times, or immune activation [44]. As an example, in preclinical models with bcl-2 mRNA melanoma cell lines, oblimersen was able to induce apoptosis. However, the poorest antiproliferative effect was observed in cells in which bcl-2 was highly expressed [133]. The reasons for the lack of correlation between oblimersen activity and bcl-2 could not be identified, but this raised the question of whether other mechanisms could be causing the antitumor activity, such as a possible immunostimulatory action or an off-target effect of the drug. Another concern relates to the hybridization-dependent toxicity, caused by exaggerated action of the drug or off-target hybridization. One example is LY2275796 targeting of elF-4E, in which the target gene together with housekeeping genes was affected. It is unclear whether the mechanism of action was mediated by sequence specificity or influenced by off-target hybridization [44,58]. Additionally, cancer is maintained by genetically independent and functionally redundant alterations [134]. Another limitation of antisense therapy may be that it affects only a single target, in opposition to the diverse and heterogeneous genetic and epigenetic alterations of each cancer [133]. Clinical responses with single agents, despite targeting, have been generally transitory and too often followed by relapse or progression [135]. It is reasonable to think that the lack of response is related to the inability of the drug to target the core mechanisms associated with an individual cancer. In a model developed for treatment of CML with targeted therapies, based on the rate of mutations, number of therapeutics, and disease volume, a minimum of three therapeutic agents seems to be required to minimize tumor resistance [136]. Accordingly, there is a rationale for combining RNA therapeutics with other treatment modalities, such as known active chemotherapy, thus exploring the synergic mechanisms of that combination. The combination of LErafAON-ETU with radiotherapy in advanced cancers or aprinocarsen with platinum-based chemotherapy in lung malignancies are some examples [85,88,137]. Several trials with different combinations are ongoing, and positive results are expected with optimism. The combination of several antisense drugs may be explored in the coming years. In the past decades, substantial development has occurred in the creation and validation of antisense therapies. At the present time, several drugs are in clinical trials, but a significant impact on the treatment of cancer is still to come. Despite the challenging obstacles, we are optimistic that emerging antisense drugs will progress through clinical development and bring new hope in the treatment of cancer. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflict of interest None. References [1] Wu SY, Lopez-Berestein G, Calin GA, Sood AK. RNAi therapies: drugging the undruggable. Sci Transl Med 2014;6:240ps247. [2] Burnett JC, Rossi JJ. RNA-based therapeutics – current progress and future prospects. Chem Biol 2012;19:60–71.

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