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.

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.

The molecular dynamics of long noncoding RNA control of transcription in PTEN and its pseudogene.

RNA has been found to interact with chromatin and modulate gene transcription. In human cells, little is known about how long noncoding RNAs (lncRNAs) interact with target loci in the context of chromatin. We find here, using the phosphatase and tensin homolog (PTEN) pseudogene as a model system, that antisense lncRNAs interact first with a 5' UTR-containing promoter-spanning transcript, which is then followed by the recruitment of DNA methyltransferase 3a (DNMT3a), ultimately resulting in the transcriptional and epigenetic control of gene expression. Moreover, we find that the lncRNA and promoter-spanning transcript interaction are based on a combination of structural and sequence components of the antisense lncRNA. These observations suggest, on the basis of this one example, that evolutionary pressures may be placed on RNA structure more so than sequence conservation. Collectively, the observations presented here suggest a much more complex and vibrant RNA regulatory world may be operative in the regulation of gene expression.

MicroRNA-190b Targets RFWD3 in Estrogen Receptor-Positive Breast Cancer.

Background: In the year 2020, breast cancer was the most common form of cancer worldwide. Roughly 70% of breast cancers are estrogen receptor-positive (ER+). MicroRNA-190b (miR-190b) has previously been reported to be upregulated in ER+ breast cancers. Previously, we have demonstrated that miR-190b is hypomethylated in ER+ breast cancers, potentially leading to its upregulation. Objectives: To further study the role of miR-190b in ER+ breast cancer and to identify its clinically relevant targets in breast cancer. Design: Patient cohort and cell line-based RNA-sequencing analysis. Methods: The Cancer Genome Atlas was used to obtain gene expression data and clinical information on patients with breast cancer. To identify messenger RNA (mRNA) targets for miR-190b, the ER+ breast cancer cell line T-47D was used to immunoprecipitate biotin-labeled miR-190b followed by RNA sequencing. Western blot was used to confirm miR-190b target. Patient survival based on miR-190b and selected target was studied using the Cancer Genome Atlas. Results: In this study, we confirm that miR-190b is overexpressed in breast cancer via differential expression analysis and show that high expression of miR-190b results in more favorable outcomes in Luminal A patients, hazard ratio (HR) = 0.29, 95% confidence interval [CI] = 0.12-0.71, P = .0063. MicroRNA-190b target analysis identified RING finger and WD repeat domain 3 (RFWD3) as one of miR-190b regulatory targets in ER+ breast cancer. Survival analysis of RFWD3 showed that elevated levels result in poorer overall survival in patients with Luminal A breast cancer (HR = 2.22, 95% CI = 1.33-3.71, P = .002). Gene ontology analysis of our sequencing results indicates that miR-190b may have a role in breast cancer development and/or tumorigenesis and that it may be a suitable tool in characterization between the ER+ subtypes, Luminal A, and Luminal B. Conclusions: We show that miR-190b targets RFWD3 in ER+ breast cancers leading to lower RFWD3 protein expression. Low levels of RFWD3 are associated with better outcomes in patients with Luminal A breast cancer but not in patients with Luminal B breast cancer. These findings provide novel insights into miR-190b role in breast cancer and that its clinical relevance is subtype specific.

MicroRNA-190b targets RFWD3 in ER-positive Breast Cancer Breast cancer is the most common diagnosed type of cancer worldwide. Most of them, or 70%, overexpressed the estrogen receptor (ER) which can be targeted with drugs. MicroRNA-190b (miR-190b) is known to be overexpressed in these types of breast cancers, and we have shown that loss of DNA methylation within the genomic region of miR-190b occurs in these ER+ cancers as well, which potentially is the cause for its overexpression. We, therefore, aimed at understanding miR-190b further. To do so, we used a technique called immunoprecipitation to capture miR-190b targets and performed RNA sequencing to identify potential targets. Of the targets, we identified RFWD3 and performed a western blot to confirm whether it was a true target. Finally, we performed survival analysis using data from the Cancer Genome Atlas to see whether RFWD3 was important for patient prognosis. In summary, we identified RFWD3 to be a target of miR-190b in ER+ breast cancers and that its expression is lower when miR-190b is elevated. We also saw that lower levels of RFWD3 are linked to better outcomes in a subgroup of ER+ breast cancers called Luminal A. These findings help in understanding miR-190b and its role in breast cancer and show that its clinical relevance is subgroup specific.

Estrogen receptor-positive breast cancer and adverse outcome in BRCA2 mutation carriers and young non-carrier patients.

Estrogen receptor-positive (ER+) breast cancer generally confers a more favorable prognosis than ER-negative cancer, however, a different picture is emerging for BRCA2 mutation carriers and young patients. We used nationwide data from population-based registries to study prognostic effects in those two groups. Of all 2817 eligible women diagnosed with breast cancer in Iceland during 1980-2004, 85% had been tested for the Icelandic 999del5 BRCA2 (c.771_775delTCAAA) founder pathogenic variant. We compared breast cancer-specific survival, effects of ER status, other clinical parameters, and treatment, between three mutually exclusive groups: BRCA2-carriers, non-carriers diagnosed 40 years or younger, and older non-carriers. Prevalence of the BRCA2 mutation among tested patients <=40 years of age was 21.0%, but it was 5.4% among women diagnosed >40 years of age. For ER+ cancer, breast cancer-specific 15-year survival was 49.7%, 55.2%, and 74.7%, among BRCA2-carriers, young and older non-carriers, respectively, whereas for ER-negative cancer, survival was similar (64.0-69.3%) for all three groups. Neither BRCA2 carriers nor young non-carriers did tumor grade 3 predict worse survival than did tumor grade 1. The adverse outcome for the young cases cannot be explained by BRCA2 mutations, as carriers were excluded from the group. Those two clinically important patient groups need special attention with respect to treatment choices, in particular, if diagnosed with ER+ tumors. It is thus advisable to have knowledge of BRCA2 status when treatment decisions are made. Finally, it is important to understand the biological basis for the specific nature of ER+ tumors in young women and BRCA2 carriers.

Sensitivity of BRCA2 mutated human cell lines to Aurora kinase inhibition.

Aurora kinases play a vital part in successful mitosis and cell division. Aberrant Aurora-A and -B expression is commonly seen in various types of tumors. Small molecule Aurora inhibitors have already entered clinical trials. Aurora-A amplification has been shown to be associated with breast tumors from BRCA2-mutation carriers and such patients might therefore be candidates for treatment with Aurora kinase inhibitors. There is a need to identify markers that can predict sensitivity to Aurora inhibition. In this study sensitivity to the inhibitor ZM447439 was tested on a panel of 15 non-malignant and malignant epithelial cell lines that differed with respect to BRCA2 and p53 status and related to level of Aurora kinase expression. The IC(50) value for cell survival ranged from 1.9-8.1 µM and was not related to presence or absence of BRCA2 mutation. The levels of Aurora-A and -B expression correlated with each other but sensitivity towards ZM447439 did not correlate with levels of Aurora-A and -B mRNA expression, alone. Cells treated with the Aurora kinase inhibitor completed mitosis but cytokinesis was inhibited resulting in polyploidy and multinucleation. Different levels of polyploidy could not be fully explained by defects in p53. Only cell lines with a combination of high Aurora-A and -B expression, BRCA2 mutation and p53 defects showed more sensitivity towards Aurora inhibition than other cell lines. In conclusion, BRCA2-mutated cells showed variable sensitivity towards Aurora kinase inhibition. The level of sensitivity could not be predicted by Aurora expression levels alone but BRCA2 mutated tumors with high Aurora expression and non-functional p53 are likely candidates for treatment with Aurora inhibitors.

PTENP1-AS contributes to BRAF inhibitor resistance and is associated with adverse clinical outcome in stage III melanoma.

BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma.

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.

Silencing of CEBPB-AS1 modulates CEBPB expression and resensitizes BRAF-inhibitor resistant melanoma cells to vemurafenib.

Introduction of targeted therapy in the treatment of metastatic cutaneous malignant melanoma (CMM) has improved clinical outcome during the last years. However, only in a subset of the CMM patients, this will lead to long-term effects. CEBPB is a transcription factor that has been implicated in various physiological and pathological processes, including cancer development. We have investigated its prognostic impact on CMM and unexpectedly found that higher CEBPB mRNA levels correlated with a longer overall survival. Furthermore, in a small cohort of patients with metastatic CMM treated with BRAF-inhibitors, higher levels of CEBPB mRNA expression in the tumor cells prior treatment correlated to a longer progression-free survival. We have characterized an overlapping antisense transcript, CEBPB-AS1, with the aim to investigate the regulation of CEBPB expression in CMM and its impact on BRAF-inhibitor sensitivity. We demonstrated that silencing of CEBPB-AS1 resulted in epigenetic modifications in the CEBPB promoter and in increased CEBPB mRNA and protein levels, inhibited proliferation and partially resensitized BRAF-inhibitor resistant CMM cells to this drug-induced apoptosis. Our data suggest that targeting CEBPB-AS1 may represent a valuable tool to sensitize CMM cells to the BRAF-inhibitor-based therapies.

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.

Breast cancer risk associated with AURKA 91T -->A polymorphism in relation to BRCA mutations.

In this study 759 breast cancer patients, including 9 BRCA1 and 98 BRCA2 mutation carriers, and 653 mutation-negative unaffected controls were genotyped for the AURKA 91T -->A polymorphism. Individuals homozygous for the 91A allele were found to be at increased risk of breast cancer compared to 91T homozygotes (OR=1.87; 95% CI=1.09-3.21). This association was strengthened when cases carrying BRCA mutations were excluded (OR=2.00; 95% CI=1.15-3.47). BRCA carrier cases differed from sporadic cases and their allele distribution was very similar to controls. These results show a statistically significant increased risk of sporadic breast cancer for individuals that are homozygous for the 91A allele but no effect in carriers of BRCA mutations. This may throw light on previously conflicting results.

A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells.

PTEN is a tumor-suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1-encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, α and ß. The α isoform functions in trans, localizes to the PTEN promoter and epigenetically modulates PTEN transcription by the recruitment of DNA methyltransferase 3a and Enhancer of Zeste. In contrast, the ß isoform interacts with PTENpg1 through an RNA-RNA pairing interaction, which affects PTEN protein output through changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell-cycle arrest and sensitizes cells to doxorubicin, which suggests a biological function for the respective PTENpg1 expressed asRNAs.

Prostate cancer progression and survival in BRCA2 mutation carriers.

BACKGROUND: Mutations in the BRCA2 gene are associated with an increased risk of prostate cancer, but it is not known whether they are associated with progression of the disease. We compared prostate cancer-specific survival, disease stage, and tumor grade between prostate cancer patients carrying the Icelandic BRCA2 999del5 founder mutation and noncarriers. METHODS: Using population-based registries, we identified all 596 prostate cancer patients who were diagnosed in Iceland during 1955 through 2004 among 29603 male relatives of unselected breast cancer probands. BRCA2 mutation status could be determined for 527 patients (88.4%). Stage and grade were abstracted from original records, blindly with respect to mutation status, for a subgroup of 89 patients that included all mutation carriers and, for each carrier, two control patients without the BRCA2 999del5 mutation who were matched to the carrier on years of diagnosis and birth. Hazard ratios (HRs) and 95% confidence intervals (CIs) for prostate cancer-specific survival were estimated using multivariable regression models. All statistical tests were two-sided. RESULTS: The mutation was carried by 30 patients (5.7%). Compared with noncarriers, BRCA2 999del5 mutation carriers had a lower mean age at diagnosis (69.0 years versus 74.0 years; P = .002), more advanced tumor stage (stages 3 or 4, 79.3% versus 38.6%; P < .001), higher tumor grade (grades G3-4, 84.0% versus 52.7%, P = .007), and shorter median survival time (2.1 years, 95% CI = 1.4 to 3.6 years, versus 12.4 years, 95% CI = 9.9 to 19.7 years). Carrying the BRCA2 999del5 mutation was also associated with an increased risk of dying from prostate cancer (adjusting for year of diagnosis and birth, HR = 3.42, 95% CI = 2.12 to 5.51); the association remained after adjustment for stage and grade (HR = 2.35, 95% CI = 1.08 to 5.11). The prognosis of BRCA2 999del5 mutation carriers was not associated with period of diagnosis or with relatedness to breast cancer probands. CONCLUSIONS: The Icelandic BRCA2 999del5 founder mutation was strongly associated with rapidly progressing lethal prostate cancer.