Thermal proteome profiling identifies PIP4K2A and ZADH2 as off-targets of Polo-like kinase 1 inhibitor volasertib.

Polo-like kinase 1 (PLK1) is an important cell cycle kinase and an attractive target for anticancer treatments. An ATP-competitive small molecular PLK1 inhibitor, volasertib, has reached phase III in clinical trials in patients with refractory acute myeloid leukemia as a combination treatment with cytarabine. However, severe side effects limited its use. The origin of the side effects is unclear and might be due to insufficient specificity of the drug. Thus, identifying potential off-targets to volasertib is important for future clinical trials and for the development of more specific drugs. In this study, we used thermal proteome profiling (TPP) to identify proteome-wide targets of volasertib. Apart from PLK1 and proteins regulated by PLK1, we identified about 200 potential volasertib off-targets. Comparison of this result with the mass-spectrometry analysis of volasertib-treated cells showed that phosphatidylinositol phosphate and prostaglandin metabolism pathways are affected by volasertib. We confirmed that PIP4K2A and ZADH2-marker proteins for these pathways-are, indeed, stabilized by volasertib. PIP4K2A, however, was not affected by another PLK1 inhibitor onvansertib, suggesting that PIP4K2A is a true off-target of volasertib. Inhibition of these proteins is known to impact both the immune response and fatty acid metabolism and could explain some of the side effects seen in volasertib-treated patients.

RNAi prodrugs decrease elevated mRNA levels of Polo-like kinase 1 in ex vivo cultured primary cells from pediatric acute myeloid leukemia patients.

Polo-like kinase 1 (Plk1) is an important regulator of the cell cycle and it is frequently overexpressed in cancer cells. Several small molecule inhibitors have been developed to target Plk1 and some of them have reached clinical trials in adults with acute myeloid leukemia (AML). Pediatric AML patients have a poor prognosis and survivors suffer from long-term side effects. As adult AML cells have an elevated expression of Plk1, AML is a disease candidate for Plk1 inhibition. However, the relative success of clinical trials have been hampered by adverse reactions. Herein, PLK1-targeting RNA interference (RNAi) prodrugs that enter cells without a transfection reagent are used to target PLK1 selectively in primary cells from pediatric AML patients. We show that PLK1 and PLK4 mRNA expression are significantly higher in pediatric AML patients when compared to healthy donors and that PLK1 is downregulated by on average 50% using RNAi prodrugs without a significant effect on other PLK family members. In addition, the RNAi prodrug-induced decrease in PLK1 can be used to potentiate the effect of cytarabine. In summary, PLK1-targeting RNAi prodrugs can decrease the elevated levels of PLK1 in primary cells from pediatric AML patients and sensitize pediatric AML cells to chemotherapeutics.

PLK1 as a cooperating partner for BCL2-mediated antiapoptotic program in leukemia.

The deregulation of BCL2 family proteins plays a crucial role in leukemia development. Therefore, pharmacological inhibition of this family of proteins is becoming a prevalent treatment method. However, due to the emergence of primary and acquired resistance, efficacy is compromised in clinical or preclinical settings. We developed a drug sensitivity prediction model utilizing a deep tabular learning algorithm for the assessment of venetoclax sensitivity in T-cell acute lymphoblastic leukemia (T-ALL) patient samples. Through analysis of predicted venetoclax-sensitive and resistant samples, PLK1 was identified as a cooperating partner for the BCL2-mediated antiapoptotic program. This finding was substantiated by additional data obtained through phosphoproteomics and high-throughput kinase screening. Concurrent treatment using venetoclax with PLK1-specific inhibitors and PLK1 knockdown demonstrated a greater therapeutic effect on T-ALL cell lines, patient-derived xenografts, and engrafted mice compared with using each treatment separately. Mechanistically, the attenuation of PLK1 enhanced BCL2 inhibitor sensitivity through upregulation of BCL2L13 and PMAIP1 expression. Collectively, these findings underscore the dependency of T-ALL on PLK1 and postulate a plausible regulatory mechanism.

Protein Delivery by PTDs/CPPs.

The ability to deliver or transduce proteins into cells allows for the manipulation of cell biology in culture, preclinical models, and potentially human disease. Fusion proteins containing the TAT peptide transduction domain (PTD), also known as cell-penetrating peptide (CPP), allow for delivery of a wide variety of proteins, including enzymes, transcription factors, tumor suppressor proteins, and many more. TAT-fusion proteins are generated cloning in-frame into the pTAT-HA plasmid, then transformed into E. coli for expression, and purified by the 6-His affinity tag over Ni-NTA column, followed by a final IEX FPLC purification step.

The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake.

Although cell-penetrating peptides are able to deliver cargo into cells, their uptake mechanism is still not fully understood and needs to be elucidated to improve their delivery efficiency. Herein, we present evidence of a new mechanism involved in uptake, the membrane repair response. Recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. The direct penetration of hydrophilic peptides through the hydrophobic plasma membrane, however, is highly controversial. Three proteins involved in target cell apoptosis--perforin, granulysin, and granzymes--share many features common in uptake of cell-penetrating peptides (e.g., they bind proteoglycans). During perforin uptake, the protein activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, because of extracellular calcium influx. On activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane, resealing it within seconds. In this study, we have used flow cytometry, fluorescence, and electron microscopy, together with high-performance liquid chromatography and mass spectrometry, to present evidence that the membrane repair response is able to mask damages caused during cell-penetrating peptide uptake, thus preventing leakage of endogenous molecules out of the cell.

Targeting Plk1 with siRNNs in primary cells from pediatric B-cell acute lymphoblastic leukemia patients.

B-cell acute lymphoblastic leukemia (B-ALL) accounts for nearly one fifth of all childhood cancers and current challenges in B-ALL treatment include resistance, relapse and late-onset side effects due to the chemotherapy. To overcome these hurdles, novel therapies need to be investigated. One promising target is Polo-like kinase 1 (Plk1), a key regulator of the cell cycle. In this study, the Plk family expression is investigated in primary peripheral blood and bone marrow mononuclear cells from ten pediatric B-ALL patients. For the first time, short interfering RiboNucleic Neutrals (siRNNs) that enter cells without a transfection reagent are used to target Plk1 mRNA in primary cells from pediatric B-ALL patients. Our results show that the expression of Plk1 and Plk4 is significantly higher in pediatric B-ALL patients compared to healthy donors. Moreover, treatment of primary peripheral blood and bone marrow mononuclear cells from pediatric B-ALL patients, cultured ex vivo, with Plk1-targeting siRNNs results in cleavage of Plk1 mRNA. Importantly, the Plk1 knockdown is specific and does not affect other Plk members in contrast to many small molecule Plk1 inhibitors. Thus, Plk1 is a potential therapeutic target in pediatric B-ALL and selective targeting of Plk1 can be achieved by the use of siRNNs.

Designing siRNA and Evaluating Its Effect on RNA Targets Using qPCR and Western Blot.

The discovery of the RNA interference (RNAi) pathway followed by the usage of synthetic short-interfering RNAs (siRNA) has contributed greatly to the understanding of gene function. Carefully designed siRNAs can considerably improve siRNA specificity leading to more accurate and efficient gene silencing. Evaluation of gene knockdown is vital for optimization of siRNA efficacy. Here we describe the fundamental principles of siRNA design and strategies for evaluating gene knockdown.

Polo-like kinases and acute leukemia.

Acute leukemia is a common malignancy among children and adults worldwide and many patients suffer from chronic health issues using current therapeutic approaches. Therefore, there is a great need for the development of novel and more specific therapies with fewer side effects. The family of Polo-like kinases (Plks) is a group of five serine/threonine kinases that play an important role in cell cycle regulation and are critical targets for therapeutic invention. Plk1 and Plk4 are novel targets for cancer therapy as leukemic cells often express higher levels than normal cells. In contrast, Plk2 and Plk3 are considered to be tumor suppressors. Several small molecule inhibitors have been developed for targeting Plk1 inhibition. Despite reaching phase III clinical trials, one of the ATP-competitive Plk1 inhibitor, volasertib, did not induce an objective clinical response and even caused lethal side effects in some patients. In order to improve the specificity of the Plk1 inhibitors and reduce off-target side effects, novel RNA interference (RNAi)-based therapies have been developed. In this review, we summarize the mechanisms of action of the Plk family members in acute leukemia, describe preclinical studies and clinical trials involving Plk-targeting drugs and discuss novel approaches in Plk targeting.

STAT3 is activated in multicellular spheroids of colon carcinoma cells and mediates expression of IRF9 and interferon stimulated genes.

Three-dimensional cell cultures, such as multicellular spheroids (MCS), reflect the in vivo architecture of solid tumours and multicellular drug resistance. We previously identified interferon regulatory factor 9 (IRF9) to be responsible for the up-regulation of a subset of interferon (IFN)-stimulated genes (ISGs) in MCS of colon carcinoma cells. This set of ISGs closely resembled a previously identified IFN-related DNA-damage resistance signature (IRDS) that was correlated to resistance to chemo- and radiotherapy. In this study we found that transcription factor STAT3 is activated upstream of IRF9 and binds to the IRF9 promoter in MCS of HCT116 colorectal carcinoma cells. Transferring conditioned media (CM) from high cell density conditions to non-confluent cells resulted in STAT3 activation and increased expression of IRF9 and a panel of IRDS genes, also observed in MCS, suggesting the involvement of a soluble factor. Furthermore, we identified gp130/JAK signalling to be responsible for STAT3 activation, IRF9, and IRDS gene expression in MCS and by CM. Our data suggests a novel mechanism where STAT3 is activated in high cell density conditions resulting in increased expression of IRF9 and, in turn, IRDS genes, underlining a mechanism by which drug resistance is regulated.

RNAi prodrugs targeting Plk1 induce specific gene silencing in primary cells from pediatric T-acute lymphoblastic leukemia patients.

Epidemiological studies of childhood leukemia survivors reveal an alarmingly high incidence of chronic health disabilities after treatment, therefore, more specific therapies need to be developed. Polo-like kinase 1 (Plk1) is a key player in mitosis and a target for drug development as it is upregulated in multiple cancer types. Small molecules targeting Plk1 are mainly ATP-competitors and, therefore, are known to elicit side effects due to lack of specificity. RNA interference (RNAi) is known for its high catalytic activity and target selectivity; however, the biggest barrier for its introduction into clinical use is its delivery. RNAi prodrugs are modified, self-delivering short interfering Ribonucleic Neutrals (siRNNs), cleaved by cytoplasmic enzymes into short interfering Ribonucleic Acids (siRNAs) once inside cells. In this study we aimed to investigate the potential of siRNNs as therapeutic tools in T-acute lymphoblastic leukemia (T-ALL) using T-ALL cell lines and patient-derived samples. We demonstrate for the first time that RNAi prodrugs (siRNNs) targeting Plk1, can enter pediatric T-ALL patient cells without a transfection reagent and induce Plk1 knockdown on both protein and mRNA levels resulting in G2/M-arrest and apoptosis. We also show that siRNNs targeting Plk1 generate less toxicity in normal cells compared to the small molecule Plk1 inhibitor, BI6727, suggesting a potentially good therapeutic index.

Induction of RNAi Responses by Short Left-Handed Hairpin RNAi Triggers.

Small double-stranded, left-handed hairpin (LHP) RNAs containing a 5'-guide-loop-passenger-3' structure induce RNAi responses by a poorly understood mechanism. To explore LHPs, we synthesized fully 2'-modified LHP RNAs targeting multiple genes and found all to induce robust RNAi responses. Deletion of the loop and nucleotides at the 5'-end of the equivalent passenger strand resulted in a smaller LHP that still induced strong RNAi responses. Surprisingly, progressive deletion of up to 10 nucleotides from the 3'-end of the guide strand resulted in a 32mer LHP capable of inducing robust RNAi responses. However, further guide strand deletion inhibited LHP activity, thereby defining the minimal length guide targeting length to 13 nucleotides. To dissect LHP processing, we examined LHP species that coimmunoprecipitated with Argonaute 2 (Ago2), the catalytic core of RNA-induced silencing complex, and found that the Ago2-associated processed LHP species was of a length that correlated with Ago2 cleavage of the passenger strand. Placement of a blocking 2'-OMe blocking modification at the LHP predicted Ago2 cleavage site resulted in an intact LHP loaded into Ago2 and no RNAi response. Taken together, these data argue that in the absence of a substantial loop, this novel class of small LHP RNAs enters the RNAi pathway by a Dicer-independent mechanism that involves Ago2 cleavage and results in an extended guide strand. This work establishes LHPs as an alternative RNAi trigger that can be produced from a single synthesis for potential use as an RNAi therapeutic.

Identification of novel small molecules that inhibit STAT3-dependent transcription and function.

Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z' = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.

Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications.

RNA interference (RNAi) has great potential to treat human disease. However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics.

Calcium and membrane repair.

As more and more studies utilize cell-penetrating peptides to deliver pharmacologically interesting substances, there is a growing need to understand their effect on the plasma membrane. If a cell-penetrating peptide together with its cargo is to be used as a drug, it is necessary to understand how the conjugate interacts with the plasma membrane to enter the cell. A key regulator of the transportation network in the cell is calcium. This chapter describes five methods that can be employed for understanding how the plasma membrane reacts to the presence of cell-penetrating peptides and the involvement of calcium.

PTD-DRBD siRNA delivery.

A major hurdle in drug delivery today is for the drug to reach inside the cell to exert its biological effect. Many drug candidates are hydrophilic and are therefore not able to cross the hydrophobic plasma membrane, which serves to protect the cell from foreign molecules and pathogens. One promising drug candidate is the hydrophilic and negatively charged short-interfering RNA (siRNA), known to degrade target mRNA 1,000-fold more efficiently than small molecule drugs. The delivery capacity of small cationic peptides called protein transduction domains or cell-penetrating peptides, suggested them to be suitable delivery vehicles for siRNA. However, it has proven troublesome to utilize the PTD-siRNA conjugates for mRNA degradation due to the characteristics of siRNA, often resulting in precipitation and aggregation. This chapter describes a recently reported delivery strategy, PTD-DRBD fusion protein siRNA delivery, where a double-stranded RNA-binding domain expressed as a fusion protein together with three TAT PTDs binds the siRNA, thus masking the negatively charged backbone and preventing aggregation. This new protocol results in noncytotoxic mRNA degradation even more effective than lipofection.

Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides.

In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration.

A new rapid cell-penetrating peptide based strategy to produce bacterial ghosts for plasmid delivery.

The production of bacterial ghosts involves the lysis gene E plasmid in order to lyse and empty the bacteria of their cytoplasmic contents. After lysis the ghosts can either be loaded with new desired DNA and used for delivery to mammalian cells or used in vaccination. Cell-penetrating peptides have been used as delivery vehicles of drugs and oligonucleotides. Although many of them show low toxicity they have been compared to antimicrobial peptides involved in innate immunity. Recently we showed that cell-penetrating peptides also could be antimicrobial. In this study we take advantage of the antimicrobial effect of one cell-penetrating peptide, namely MAP, which is a model amphipathic peptide and treat bacteria with the peptide to produce bacterial ghosts. This new peptide based strategy is not dependent on the lysis gene E plasmid thus; several tiresome steps are removed in the production of ghosts. In addition the ghosts can be preloaded with a desired plasmid or DNA further removing time consuming reprocessing steps. To our knowledge this is the first study that uses a cell-penetrating peptide based strategy to produce bacterial ghosts to be used in plasmid delivery.