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A select group of patients with hepatocellular carcinomas (HCC) benefit from surgical, radiologic, and systemic therapies that include a combination of anti-angiogenic and immune-checkpoint inhibitors. However, because HCC is generally asymptomatic in its early stages, this not only leads to late diagnosis, but also to therapy resistance. The nucleoside analogue 6-thio-dG (THIO) is a first-in-class telomerase-mediated telomere-targeting anticancer agent. In telomerase expressing cancer cells, THIO is converted into the corresponding 5′-triphosphate, which is efficiently incorporated into telomeres by telomerase, activating telomere damage responses and apoptotic pathways. Here, we show how THIO is effective in controlling tumor growth and, when combined with immune checkpoint inhibitors, is even more effective in a T-cell-dependent manner. We also show telomere stress induced by THIO increases both innate sensing and adaptive antitumor immunity in HCC. Importantly, the extracellular high-mobility group box 1 protein acts as a prototypical endogenous DAMP (Damage Associated Molecular Pattern) in eliciting adaptive immunity by THIO. These results provide a strong rationale for combining telomere-targeted therapy with immunotherapy.

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Liver, renal spleen and colon effect.

Telomeres and IC50s.

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

Background: The modified nucleoside 6-thio-2'-deoxyguanosine (THIO) is a first-in-class direct telomere-targeting agent that is preferentially incorporated in telomeres of telomerase-positive cells, leading to telomere uncapping and cancer cell death. Preclinical evidence indicates that THIO pretreatment sensitizes non-small cell lung cancer (NSCLC) cells to cemiplimab, a PD-1 inhibitor approved as 1L treatment for patients with locally advanced/metastatic NSCLC with ≥50% PD-L1 expression. The THIO-101 trial is designed to evaluate safety and efficacy of THIO followed by cemiplimab in patients with advanced NSCLC who have developed resistance or relapsed after prior immune checkpoint inhibitor (ICI) therapy. Trial Design: THIO-101 enrolls adult patients with stage 3/4 NSCLC, who have progressed or relapsed after treatment with an ICI alone or in combination with platinum-based therapy. Using a 3+3 design, the safety lead-in (Part A) is enrolling up to 6 patients in the first cohort to receive a total dose of THIO 360 mg IV (120 mg QD, D1-3) followed by a fixed dose of cemiplimab 350 mg on D5 Q3W. If ≥2 patients experience dose-limiting toxicities, a second cohort will receive a total dose of THIO 180 mg IV (60 mg QD, D1-3) followed by cemiplimab. In the dose- finding portion of the study (Part B), patients will be randomized 1:1:1 in a Simon 2-stage design (n=41 per arm) to receive THIO 360 mg [if cleared in Part A], 180 mg, or 60 mg followed by cemiplimab. Sequential THIO and cemiplimab treatment may be continued Q3W for up to 1 year, or until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoints are safety, objective response rate, and disease control rate (CR, PR, and SD). Secondary endpoints include duration of response, progression-free survival, and overall survival; exploratory endpoints include PK/PD parameters (type I IFN, IL-6, CRP) and assessment of tumor telomerase status by IHC. Investigators will assess disease progression per RECIST v1.1 and/or iRECIST, with radiographic scans performed on D1 of cycles 3 and 5 and every 9-12 weeks thereafter. Adverse events are evaluated according to NCI CTCAE v5.0. The trial is enrolling patients at sites in Europe and Australia. The trial has completed enrollment in Part A without DLTs and opened enrollment in Part B. NCT05208944; EUDRA CT Number, 2021-005136-34 Citation Format: Mihail Obrocea, Brenton Seidl, Rohit Joshi, Melissa Moore, Vlad Vitoc, Bin Yao, Sergei Gryaznov. A phase 2, multi-center, open-label, dose-finding study evaluating telomere targeting agent THIO sequenced with cemiplimab in patients with advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT050.

SUMMARYLong noncoding RNAs (lncRNA) can drive the tumorigenesis and be susceptible to therapeutic intervention. To define the landscape of therapeutically actionable lncRNA dependencies in multiple myeloma (MM), we coupled our extensive lncRNA transcriptomic profile with lncRNA targeted CRISPR interference viability screen and identified RNA Regulator of Lipogenesis (RROL) as a leading lncRNA dependency in MM. RROL shares its origin with the microRNA locus MIR17HG, however supports the proliferation and survival of MM cells in a microRNA- and DROSHA- independent manner. We found that RROL provides a chromatin scaffold for the functional interaction between c-MYC and WDR82 to promote the regulation of the lipogenic pathways via the transcriptional control of the rate-limiting enzyme ACC1 in MM cells. Inhibition of RROL with clinically applicable antisense molecules disrupts its transcriptional and functional activities causing potent anti-tumor effects both in vitro and in vivo in two pre-clinical animal models. This study establishes lncRNA RROL as a therapeutically actionable dependency with a unique mechanism of action in support of myeloma cell growth.

Pneumocystis carinii is the most common lethal opportunistic pathogen infecting Acquired Immune Deficiency Syndrome (AIDS) patients, and more effective therapeutics for it are needed. P. carinii, but not humans, contain RNA self-splicing group I introns, so these functionally important RNAs are potential anti-fungal targets. In vitro, d(ATGACT), which mimics the 3' end of the 5' exon of a conserved ribosomal RNA group I intron from mouse-derived Pneumocystis carinii binds to a ribozyme that is a truncated form of this intron. The binding is about 30,000 times tighter than expected for simple base-pairing because binding is enhanced by tertiary interactions. Here we report the effects of modifying the phosphodiester backbone of d(ATGACT) with phosphorothioate and of d(ATGAC)rU with N3'-->P5' phosphoramidate linkages. The enhancement of binding by tertiary interactions is not substantially decreased, and in some cases is increased when single Rp and Sp phosphorothioate substitutions are made, although overall binding is weaker by up to 6-fold. A mixture of 5' exon mimic isomers that each contain five phosphorothioate linkages binds to the ribozyme at least 14-fold less tightly than the corresponding phosphodiester mimic. In contrast, the 5' exon mimic with five internal N3'-->P5' phosphoramidate linkages binds 4-fold more tightly than d(ATGAC)rU. This increased binding is largely due to more favorable base-pairing, but tertiary interactions still enhance binding by more than 2, 000-fold. These results indicate that chemically modified, nuclease stable 5' exon mimics can act as antisense agents with binding enhanced by tertiary interactions (BETI). This strategy permits design of short antisense agents with high specificity.

Telomeres are specialized nucleoprotein complexes that protect against fusion and degradation of linear chromosomes. Critical shortening of telomeres leads to irreversible cessation of cell division, whereas telomerase elongates telomere sequences to compensate for losses that occur with each round of DNA replication. Continued proliferation of tumor cells requires this enzyme to maintain chromosomal stability and to counteract the cellular mitotic clock. In this study, we evaluated the effect of oligonucleotide N3'--P5' thio-phosphoramidate (NP), which targets template RNA component, in human multiple myeloma (MM) cell lines and patient MM cells. Fluorescein staining at 24 h confirmed NP uptake in 84.7 and 86.1% of MM.1S cells and MM patient cells, respectively, without any transfection enhancer. High transfection efficiency was observed into both CD138(+) and CD138(-) MM patient cells. Match NP (7S), but not mismatch NP (30S), inhibited telomerase activity in MM.1S cells, U266 cells, and RPMI 8226 cells, as well as in patient MM cells. Moreover, 7S inhibited cytokine-induced telomerase activity in MM.1S cells. 7S treatment-induced progressive telomere shortening was associated with growth inhibition and cell death in MM.1S cells with short telomeres (2.5 kb), but not in U266 cells with long telomeres (9.0 kb), at 56 days of culture. Progressive telomere shortening leading to growth inhibition and cell death in MM.1S cells was associated with up-regulation of p21 and phosphorylation of p53 (Ser-15). These studies, therefore, identify the molecular sequelae of NP oligonucleotide (GRN163) against human telomerase RNA component as a telomerase inhibitor and provide the rationale for the development of telomerase-targeted therapies to improve patient outcome in MM.

The effects of telomerase inhibition with an oligonucleotide N3′ → P5′ thiophosphoramidate (GRN163) complementary to the telomerase template region were examined on human multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) cell lines, primary MM cells, and tumor xenografts. GRN163 treatment reduced telomerase levels in all cells and induced more rapid telomeric shortening. Continuous GRN163 treatment for 7 to 14 days resulted in proliferative arrest, morphologic changes, and apoptosis characteristic of cell crisis in tumor cell lines with short (1.7-5.4 kb) but not long (9-11 kb) telomeres. Intratumoral administration of GRN163 also inhibited the growth of MM and NHL xenografts established from cell lines with short telomeres (Hs602 lymphoma, 2.7 kb; CAG myeloma, 2.7 kb) and increased tumor apoptosis. However, GRN163 therapy of NHL xenografts established from cells with long telomeres (11.0 kb) had equivocal effects on tumor growth and did not induce apoptosis during this time frame. Systemic daily intraperitoneal administration of GRN163 in myeloma xenografts with short telomere lengths also decreased tumor telomerase levels and reduced tumor volumes. These data demonstrate that telomerase is important for the replication of mature B-cell neoplasia by stabilizing short telomeres, and they suggest that telomerase inhibition represents a novel therapeutic approach to MM and NHL.

Telomerase, the enzyme responsible for proliferative immortality, is expressed in essentially all cancer cells, but not in most normal human cells. Thus, specific telomerase inhibition is potentially a universal anticancer therapy with few side effects. We designed N3'--P5' thio-phosphoramidate (NPS) oligonucleotides as telomerase template antagonists and found that their ability to form stable duplexes with the telomerase RNA subunit was the key factor for antitelomerase activity. In biochemical assays 11-13-mer NPS oligonucleotides demonstrated sequence- and dose-dependent inhibition of telomerase with IC(50) values1 nM. Optimization of the sequence, length, and bioavailability resulted in the selection of a 13-mer NPS oligonucleotide, GRN163, as a drug development candidate. GRN163 inhibited telomerase in a cell-free assay at 45 +/- 7 pM, and in various tumor cell lines at approximately 1 nM and approximately 0.3-1.0 micro M in the presence and absence of carriers, respectively. GRN163 was competitive with telomeric primer binding, primarily because of hybridization to human telomerase RNA (hTR) component. Tumor cells treated with GRN163 in culture underwent telomere shortening, followed by cellular senescence or apoptosis after a period of time that generally correlated with initial telomere length. In a flank DU145 (prostate cancer) xenograft model, parenterally administered GRN163 caused suppression of tumor growth in the absence of gross toxicity. These data demonstrate that GRN163 has significant potential for additional development as an anticancer agent.

Candida albicans is one of many infectious pathogens that are evolving resistance to current treatments. RNAs provide a large class of targets for new therapeutics for fighting these organisms. One strategy for targeting RNAs uses short oligonucleotides that exhibit binding enhancement by tertiary interactions in addition to Watson-Crick pairing. A potential RNA target in C. albicans is the self-splicing group I intron in the LSU rRNA precursor. The recognition elements that align the 5' exon splice site for a ribozyme derived from this precursor are complex [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. These recognition elements have been used to guide design of hexanucleotide mimics of the 5' exon that have backbones modified for nuclease stability. These hexanucleotides bind as much as 100000-fold more tightly to a ribozyme derived from the intron than to a hexanucleotide mimic of the intron's internal guide sequence, r(GGAGGC). Several of these oligonucleotides inhibit precursor self-splicing via a suicide inhibition mechanism. The most promising suicide inhibitor is the ribophosphoramidate rn(GCCUC)rU, which forms more trans-spliced than cis-spliced product at oligonucleotide concentrations of >100 nM at 1 mM Mg(2+). The results indicate that short oligonucleotides modified for nuclease stability can target catalytic RNAs when the elements of tertiary interactions are complex.

Pneumocystis carinii is a mammalian pathogen that infects and kills immunocompromised hosts such as cancer and AIDS patients. The LSU rRNA precursor of P. carinii contains a conserved group I intron that is an attractive drug target because humans do not contain group I introns. The oligonucleotide r(AUGACU), whose sequence mimics the 3'-end of the 5'-exon, binds to a ribozyme derived from the intron with a K(d) of 5.2 nM, which is 61000-fold tighter than expected from base-pairing alone [Testa, S. M., Haidaris, G. C., Gigliotti, F., and Turner, D. H. (1997) Biochemistry 36, 9379-9385]. Thus, oligonucleotide binding is enhanced by tertiary interactions. To localize interactions that give rise to this tertiary stability, binding to the ribozyme has been measured as a function of oligonucleotide length and sequence. The results indicate that 4.3 kcal/mol of tertiary stability is due to a G.U pair that forms at the intron's splice junction. Eliminating nucleotides at the 5'-end of r(AUGACU) does not affect intron binding more than expected from differences in base-pairing until r((___)ACU), which binds much more tightly than expected. Adding a C at the 5'- or 3'-end that can potentially form a C-G pair with the target has little effect on binding affinity. Truncated oligonucleotides were tested for their ability to inhibit intron self-splicing via a suicide inhibition mechanism. The tetramer, r((__)GACU), retains similar binding affinity and reactivity as the hexamer, r(AUGACU). Thus oligonucleotides as short as tetramers might serve as therapeutics that can use a suicide inhibition mechanism to inhibit self-splicing. Results with a phosphoramidate tetramer and thiophosphoramidate hexamer indicate that oligonucleotides with backbones stable to nuclease digestion retain favorable binding and reactivity properties.