Nucleic acid therapeutics as differentiation agents for myeloid leukemias

Abstract

Differentiation therapy has proven to be a success story for patients with acute promyelocytic leukemia. However, the remaining subtypes of acute myeloid leukemia (AML) are treated with cytotoxic chemotherapies that have limited efficacy and a high likelihood of resistance. As differentiation arrest is a hallmark of AML, there is increased interest in developing differentiation-inducing agents to enhance disease-free survival. Here, we provide a comprehensive review of current reports and future avenues of nucleic acid therapeutics for AML, focusing on the use of targeted nucleic acid drugs to promote differentiation. Specifically, we compare and discuss the precision of small interfering RNA, small activating RNA, antisense oligonucleotides, and aptamers to modulate gene expression patterns that drive leukemic cell differentiation. We delve into preclinical and clinical studies that demonstrate the efficacy of nucleic acid-based differentiation therapies to induce leukemic cell maturation and reduce disease burden. By directly influencing the expression of key genes involved in myeloid maturation, nucleic acid therapeutics hold the potential to induce the differentiation of leukemic cells towards a more mature and less aggressive phenotype. Furthermore, we discuss the most critical challenges associated with developing nucleic acid therapeutics for myeloid malignancies. By introducing the progress in the field and identifying future opportunities, we aim to highlight the power of nucleic acid therapeutics in reshaping the landscape of myeloid leukemia treatment.

Fig. 1. Mechanism of small interfering RNA.

RNAi is executed by the RNA-Induced Silencing Complex (RISC), a multiprotein complex with Argonaute-2 (AGO2) as the main effector protein. Following delivery of siRNA to the cytoplasm and assembly of the RISC (step 1), the short RNA duplex is loaded onto RISC (step 2) and the strands are separated (step 3), with the most energetically favorable strand being incorporated into the complex. The RISC then associates with target messenger RNA (mRNA) via complementary binding to the single-stranded siRNA (step 4), and the catalytic site of AGO2 cleaves the mRNA into small fragments (step 5). Effective mRNA degradation via RNAi can silence gene expression below detectable levels.

Fig. 2. Mechanism of antisense oligonucleotides.

A Splice-switching ASOs interfere with RNA-binding proteins and non-coding RNA that direct splicing. By preventing binding of splicing machinery to the splice site or blocking pro-splicing effects, ASOs enable entire exons to be excluded from mature mRNA. Alternatively, by blocking binding of splicing repressors, ASOs ensure inclusion of target exons in mature mRNA. B ASO-mediated inhibition of natural antisense transcripts regulates antisense transcripts produced from the antisense strand of DNA during transcription of the protein-coding gene located on the sense strand. Antisense transcriptions negatively or positively regulate the expression of their corresponding sense gene either by interacting directly with pre-mRNA or by regulation transcription of the gene itself. C RNase H1-dependent ASOs hybridize to complementary target RNA forming a DNA-RNA heteroduplex that results in RNase H1 cleavage of the RNA. D Anti-miRNA ASOs (antagomirs) sequester mature miRNA through complementary binding to their seed region (nucleotides 2 to 8 of miRNA). Such ASOs competitively inhibit the endogenous function of a target miRNA and block the ability of miRNA from either inhibiting or enhancing gene expression. E mRNA stabilizing ASOs promote mRNA stability by preventing formation of 5’-UTR secondary structures; or redirecting polyadenylation to an alternate upstream site to remove 3’-UTR destabilizing regions. F Steric block ASOs physically block translational machinery to the target mRNA. In mRNAs with multiple start codons, steric block ASOs redirect translational machinery to a secondary start codon, thereby allowing selective expression of specific protein isoforms.

Fig. 3. Aptamers as therapeutics and delivery tools.

A The SELEX process involves iterative rounds of incubating the target of interest with large oligonucleotide libraries, washing, and collecting the highest binding sequences. B Therapeutic aptamers can function as agonists or antagonists. As agonists, they induce an active conformation of the receptor through allosteric interactions. As antagonists, they physically block the binding site of the receptor, induce inactive conformations through allosteric binding, interfere with receptor dimerization, reduce cell surface receptors by initiating internalization or degradation, and/or inhibit signal transmission by binding to a messenger molecule. Aptamer-based drug delivery strategies help deliver therapeutic agents to the target site via conjugation to a specific agent minimizing off-target effects.

Fig. 4. Mechanism of small activating RNAs.

saRNAs reversibly increase mRNA expression above endogenous levels by targeting the promoter region of the gene of interest. Once the saRNA is delivered to the cytoplasm, AGO2 and heterogeneous nuclear ribonucleoproteins (hnRNPs) bind to the saRNA duplex. The guide strand is retained, and the passenger strand is discarded. Following translocation of saRNA-AGO2/hnRNPs complex to the nucleus, the guide strand will bind to a complementary sequence near the promoter, resulting in recruitment of transcription initiation and elongation factors, such as RNA polymerase II, RNA helicase A, RNA polymerase-associated protein CTR9 homolog, and RNA polymerase II-associated factor 1 homolog. At the nucleosome level, saRNAs activate transcription by loosening the chromatin through epigenetic changes and histone modifications, such as reduced acetylation and dimethylation of histone H3K9, increased di/trimethylation at histone H3K4, and monoubiquitination on histone H2B [18]. These epigenetic changes are potentially responsible for the long-lasting and sustained gene upregulation induced by saRNAs.

Fig. 5. Identification and design of NATs for myeloid leukemia targets.

This chart provides a step-by-step guide to identifying the ideal NAT for your target and then designing and screening that NAT.