Multisite Verification of a Targeted CFTR Polymerase Chain Reaction/Capillary Electrophoresis Assay That Evaluates Pathogenic Variants Across Diverse Ethnic and Ancestral Groups.

CONTEXT.­: Existing targeted cystic fibrosis screening assays miss important pathogenic CFTR variants in the ethnically diverse US population. OBJECTIVE.­: To evaluate the analytic performance of a multiplex polymerase chain reaction (PCR)/capillary electrophoresis (CE) CFTR assay panel that simultaneously interrogates primary pathogenic variants of different ethnic/ancestral groups. DESIGN.­: Performance characteristic assessment and variant coverage comparison of the panel with a focus on ethnicity-specific CFTR variants were performed. Sample DNA was primarily from whole blood or cell lines. Detection of CFTR carriers was compared across several commercially available CFTR kits and recommended variant sets based on panel content. RESULTS.­: The panel interrogated 65 pathogenic CFTR variants representing 92% coverage from a recent genomic sequencing survey of the US population, including 4 variants with top 5 frequency in African or Asian populations not reflected in other targeted panels. In simulation studies, the panel represented 95% of carriers across the global population, resulting in 6.9% to 19.0% higher carrier detection rate compared with 10 targeted panels or variant sets. Precision and sensitivity/specificity were 100% concordant. Multisite sample-level genotyping accuracy was 99.2%. Across PCR and CE instruments, sample-level genotyping accuracy was 97.1%, with greater than 99% agreement for all variant-level metrics. CONCLUSIONS.­: The CFTR assay achieves 92% or higher coverage of CFTR variants in diverse populations and provides improved pan-ethnic coverage of minority subgroups of the US populace. The assay can be completed within 5 hours from DNA sample to genotype, and performance data exceed acceptance criteria for analytic metrics. This assay panel content may help address gaps in ancestry-specific CFTR genotypes while providing a streamlined procedure with rapidly generated results.

Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours.

BACKGROUND: In this first-in-human, Phase 1 study of a microRNA-based cancer therapy, the recommended Phase 2 dose (RP2D) of MRX34, a liposomal mimic of microRNA-34a (miR-34a), was determined and evaluated in patients with advanced solid tumours. METHODS: Adults with various solid tumours refractory to standard treatments were enrolled in 3 + 3 dose-escalation cohorts and, following RP2D determination, expansion cohorts. MRX34, with oral dexamethasone premedication, was given intravenously daily for 5 days in 3-week cycles. RESULTS: Common all-cause adverse events observed in 85 patients enrolled included fever (% all grade/G3: 72/4), chills (53/14), fatigue (51/9), back/neck pain (36/5), nausea (36/1) and dyspnoea (25/4). The RP2D was 70 mg/m2 for hepatocellular carcinoma (HCC) and 93 mg/m2 for non-HCC cancers. Pharmacodynamic results showed delivery of miR-34a to tumours, and dose-dependent modulation of target gene expression in white blood cells. Three patients had PRs and 16 had SD lasting ≥4 cycles (median, 19 weeks, range, 11-55). CONCLUSION: MRX34 treatment with dexamethasone premedication demonstrated a manageable toxicity profile in most patients and some clinical activity. Although the trial was closed early due to serious immune-mediated AEs that resulted in four patient deaths, dose-dependent modulation of relevant target genes provides proof-of-concept for miRNA-based cancer therapy. CLINICAL TRIAL REGISTRATION: NCT01829971.

Synergy between next generation EGFR tyrosine kinase inhibitors and miR-34a in the inhibition of non-small cell lung cancer.

OBJECTIVES: EGFR tyrosine kinase inhibitors (TKIs) are widely used to treat NSCLC, primarily patients with activating mutations, with more limited response in wild-type disease. However, even with EGFR-mutated disease, many patients fail to respond, most who initially respond fail to respond completely, and almost all develop resistance and inevitably progress. New therapeutic options that improve these outcomes could provide substantial clinical benefit. We previously demonstrated strong synergistic effects between erlotinib and the tumor suppressor microRNA miR-34a, sensitizing NSCLC cells with primary resistance (EGFR wild-type) and restoring sensitivity in cells with acquired resistance. Here, we report results of further research combining miR-34a with newer generation EGFR-TKIs in similar experiments. MATERIALS AND METHODS: Human NSCLC cell lines with varying degrees of primary and acquired resistance to erlotinib were assessed for sensitivity to a broad set of combined doses of miR-34a mimic and afatinib, rociletinib or osimertinib. Multiple analytical approaches were used to characterize effects on cancer cell proliferation as additive, antagonistic or synergistic. RESULTS: Mimics of miR-34a synergized with afatinib, rociletinib or osimertinib in all EFGR-mutant cells tested. Best and consistently strong synergy was observed in cell models with acquired resistance. Synergy was also evident in most EGFR wild-type cells with miR-34a combined with rociletinib and osimertinib, but not with afatinib. The effects were observed across a broad range of dose levels and drug ratios, with maximal synergy at doses yielding high levels of inhibition beyond those possible to be induced by the single agents alone. CONCLUSION: Combined miR-34a and EGFR-TKIs synergistically sensitize both EGFR wild-type and mutant NSCLC cells, supporting clinical investigation of these combinations as a strategy to overcome both primary and acquired resistance to EGFR-TKIs in NSCLC, possibly with an improved therapeutic index.

PDL1 Regulation by p53 via miR-34.

BACKGROUND: Although clinical studies have shown promise for targeting PD1/PDL1 signaling in non-small cell lung cancer (NSCLC), the regulation of PDL1 expression is poorly understood. Here, we show that PDL1 is regulated by p53 via miR-34. METHODS: p53 wild-type and p53-deficient cell lines (p53(-/-) and p53(+/+) HCT116, p53-inducible H1299, and p53-knockdown H460) were used to determine if p53 regulates PDL1 via miR-34. PDL1 and miR-34a expression were analyzed in samples from patients with NSCLC and mutated p53 vs wild-type p53 tumors from The Cancer Genome Atlas for Lung Adenocarcinoma (TCGA LUAD). We confirmed that PDL1 is a direct target of miR-34 with western blotting and luciferase assays and used a p53(R172HΔ)g/+K-ras(LA1/+) syngeneic mouse model (n = 12) to deliver miR-34a-loaded liposomes (MRX34) plus radiotherapy (XRT) and assessed PDL1 expression and tumor-infiltrating lymphocytes (TILs). A two-sided t test was applied to compare the mean between different treatments. RESULTS: We found that p53 regulates PDL1 via miR-34, which directly binds to the PDL1 3' untranslated region in models of NSCLC (fold-change luciferase activity to control group, mean for miR-34a = 0.50, SD = 0.2, P < .001; mean for miR-34b = 0.52, SD = 0.2, P = .006; and mean for miR-34c = 0.59, SD = 0.14, and P = .006). Therapeutic delivery of MRX34, currently the subject of a phase I clinical trial, promoted TILs (mean of CD8 expression percentage of control group = 22.5%, SD = 1.9%; mean of CD8 expression percentage of MRX34 = 30.1%, SD = 3.7%, P = .016, n = 4) and reduced CD8(+)PD1(+) cells in vivo (mean of CD8/PD1 expression percentage of control group = 40.2%, SD = 6.2%; mean of CD8/PD1 expression percentage of MRX34 = 20.3%, SD = 5.1%, P = .001, n = 4). Further, MRX34 plus XRT increased CD8(+) cell numbers more than either therapy alone (mean of CD8 expression percentage of MRX34 plus XRT to control group = 44.2%, SD = 8.7%, P = .004, n = 4). Finally, miR-34a delivery reduced the numbers of radiation-induced macrophages (mean of F4-80 expression percentage of control group = 52.4%, SD = 1.7%; mean of F4-80 expression percentage of MRX34 = 40.1%, SD = 3.5%, P = .008, n = 4) and T-regulatory cells. CONCLUSIONS: We identified a novel mechanism by which tumor immune evasion is regulated by p53/miR-34/PDL1 axis. Our results suggest that delivery of miRNAs with standard therapies, such as XRT, may represent a novel therapeutic approach for lung cancer.

A qRT-PCR Method for Determining the Biodistribution Profile of a miR-34a Mimic.

MRX34 has recently entered the clinic as the first therapeutic product based on a microRNA (miRNA) mimic. In order to measure drug concentrations in vivo, a quantitation method is needed that exhibits high precision, accuracy, and robustness. While most clinical applications for oligonucleotide therapeutics involve methods based on hybridization assays and liquid chromatography-tandem mass spectrometry, quantitative PCR (qPCR) is a less well-described approach. Here, we present an RT, qPCR, and analysis method to determine the tissue biodistribution of endogenous as well as a therapeutic, exogenous miRNA mimic therapeutic. Assay performance is demonstrated on multiple tissues from nonhuman primates dosed with MRX34.

In-depth analysis shows synergy between erlotinib and miR-34a.

Tyrosine kinase inhibitors directed against epidermal growth factor receptor (EGFR-TKI), such as erlotinib, are effective in a limited fraction of non-small cell lung cancer (NSCLC). However, the majority of NSCLC and other cancer types remain resistant. Therapeutic miRNA mimics modeled after endogenous tumor suppressor miRNAs inhibit tumor growth by repressing multiple oncogenes at once and, therefore, may be used to augment drug sensitivity. Here, we investigated the relationship of miR-34a and erlotinib and determined the therapeutic activity of the combination in NSCLC cells with primary and acquired erlotinib resistance. The drug combination was also tested in a panel of hepatocellular carcinoma cells (HCC), a cancer type known to be refractory to erlotinib. Using multiple analytical approaches, drug-induced inhibition of cancer cell proliferation was determined to reveal additive, antagonistic or synergistic effects. Our data show a strong synergistic interaction between erlotinib and miR-34a mimics in all cancer cells tested. Synergy was observed across a range of different dose levels and drug ratios, reducing IC50 dose requirements for erlotinib and miR-34a by up to 46-fold and 13-fold, respectively. Maximal synergy was detected at dosages that provide a high level of cancer cell inhibition beyond the one that is induced by the single agents alone and, thus, is of clinical relevance. The data suggest that a majority of NSCLC and other cancers previously not suited for erlotinib may prove sensitive to the drug when used in combination with a miR-34a-based therapy.

Quantification of therapeutic miRNA mimics in whole blood from nonhuman primates.

MRX34, a microRNA (miRNA)-based therapy for cancer, has recently entered clinical trials as the first clinical candidate in its class. It is a liposomal nanoparticle loaded with a synthetic mimic of the tumor suppressor miRNA miR-34a as the active pharmaceutical ingredient. To understand the pharmacokinetic properties of the drug and to rationalize an optimal dosing regimen in the clinic, a method is needed to quantitatively detect the miRNA mimic. Here, we report the development and qualification of a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay in support of pharmacokinetic and toxicokinetic assessments in the nonhuman primate. Detection and quantification were performed on total ribonucleic acid (RNA) isolated from whole blood. The qualified range of the standard curve spans 6 orders of magnitude from 2.5 × 10(-7) to 2.5 × 10(-1) ng per reverse transcription (RT) reaction, corresponding to an estimated blood concentration from 6.2 × 10(-5) to 6.2 × 10(1) ng/mL. Our results demonstrate that endogenous as well as the exogenous miR-34a can be accurately and precisely quantified. The assay was used to establish the pharmacokinetic profile of MRX34, showing a favorable residence time and exposure of the miRNA mimic in whole blood from nonhuman primates.

Distinct microRNA expression profiles in prostate cancer stem/progenitor cells and tumor-suppressive functions of let-7.

MiRNAs regulate cancer cells, but their potential effects on cancer stem/progenitor cells are still being explored. In this study, we used quantitative real-time-PCR to define miRNA expression patterns in various stem/progenitor cell populations in prostate cancer, including CD44+, CD133+, integrin α2ß1+, and side population cells. We identified distinct and common patterns in these different tumorigenic cell subsets. Multiple tumor-suppressive miRNAs were downregulated coordinately in several prostate cancer stem/progenitor cell populations, namely, miR-34a, let-7b, miR-106a, and miR-141, whereas miR-301 and miR-452 were commonly overexpressed. The let-7 overexpression inhibited prostate cancer cell proliferation and clonal expansion in vitro and tumor regeneration in vivo. In addition, let-7 and miR-34a exerted differential inhibitory effects in prostate cancer cells, with miR-34a inducing G1 phase cell-cycle arrest accompanied by cell senescence and let-7 inducing G2-M phase cell-cycle arrest without senescence. Taken together, our findings define distinct miRNA expression patterns that coordinately regulate the tumorigenicity of prostate cancer cells.

miR-10a overexpression is associated with NPM1 mutations and MDM4 downregulation in intermediate-risk acute myeloid leukemia.

OBJECTIVE: The study investigated differential microRNA (miRNA) expression patterns in acute myeloid leukemia (AML) patients with intermediate-risk (IR) characteristics. After characterization and validation of miR-10a, which was specifically upregulated in nucleophosmin 1 (NPM1) mutant AML samples, functional consequences of miR-10a overexpression were further delineated in vitro. MATERIALS AND METHODS: Microarray analysis of miRNAs in bone marrow samples from AML (IR) patients with NPM1 mutations and healthy donors was performed to detect differential expression patterns. After validation of miRNA expression specific for NPM1 mutation in AML patients by quantitative reverse transcription polymerase chain reaction, a functional target gene search was conducted using complementary DNA microarray data from samples transfected with miR-10a. Potential target gene validation was done using transient transfection of K562 cells followed by Western blotting and luciferase reporter assay. RESULTS: In comparison with wild-type samples, NPM1 mutant AML samples were shown to markedly overexpress miR-10a. Subsequent in vitro miR-10a overexpression induced differential gene expression as determined by microarray analysis. Here the murine double minute 4 (MDM4) gene turned out as a candidate gene for miR-10a. Validation of MDM4 in leukemic cells revealed a robust negative relationship between miR-10a overexpression and MDM4 downregulation. Furthermore, we determined an inverse association between miR-10a and MDM4 expression in AML (IR) samples with respect to their NPM1 mutational status. CONCLUSIONS: miR-10a expression is highly characteristic for AML (IR) patients with NPM1 mutations and may influence its biological properties in AML by interfering with the p53 machinery partly regulated by MDM4.

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.

Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44(+) cell population, but whether miRNAs regulate CD44(+) prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44(+) prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44(+) prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44(-) prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44(+) prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.

Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34.

Tumor suppressor microRNAs (miRNA) provide a new opportunity to treat cancer. This approach, "miRNA replacement therapy," is based on the concept that the reintroduction of miRNAs depleted in cancer cells reactivates cellular pathways that drive a therapeutic response. Here, we describe the development of a therapeutic formulation using chemically synthesized miR-34a and a lipid-based delivery vehicle that blocks tumor growth in mouse models of non-small-cell lung cancer. This formulation is effective when administered locally or systemically. The antioncogenic effects are accompanied by an accumulation of miR-34a in the tumor tissue and downregulation of direct miR-34a targets. Intravenous delivery of formulated miR-34a does not induce an elevation of cytokines or liver and kidney enzymes in serum, suggesting that the formulation is well tolerated and does not induce an immune response. The data provide proof of concept for the systemic delivery of a synthetic tumor suppressor mimic, obviating obstacles associated with viral-based miRNA delivery and facilitating a rapid route for miRNA replacement therapy into the clinic.

Systemic delivery of synthetic microRNA-16 inhibits the growth of metastatic prostate tumors via downregulation of multiple cell-cycle genes.

Recent reports have linked the expression of specific microRNAs (miRNAs) with tumorigenesis and metastasis. Here, we show that microRNA (miR)-16, which is expressed at lower levels in prostate cancer cells, affects the proliferation of human prostate cancer cell lines both in vitro and in vivo. Transient transfection with synthetic miR-16 significantly reduced cell proliferation of 22Rv1, Du145, PPC-1, and PC-3M-luc cells. A prostate cancer xenograft model revealed that atelocollagen could efficiently deliver synthetic miR-16 to tumor cells on bone tissues in mice when injected into tail veins. In the therapeutic bone metastasis model, injection of miR-16 with atelocollagen via tail vein significantly inhibited the growth of prostate tumors in bone. Cell model studies indicate that miR-16 likely suppresses prostate tumor growth by regulating the expression of genes such as CDK1 and CDK2 associated with cell-cycle control and cellular proliferation. There is a trend toward lower miR-16 expression in human prostate tumors versus normal prostate tissues. Thus, this study indicates the therapeutic potential of miRNA in an animal model of cancer metastasis with systemic miRNA injection and suggest that systemic delivery of miR-16 could be used to treat patients with advanced prostate cancer.

Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways.

MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurodegenerative diseases such as Alzheimer's disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

Genome-scale microRNA and small interfering RNA screens identify small RNA modulators of TRAIL-induced apoptosis pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4/5 and selectively induces caspase-dependent apoptosis. The RNA interference screening approach has led to the discovery and characterization of several TRAIL pathway components in human cells. Here, libraries of synthetic small interfering RNA (siRNA) and microRNAs (miRNA) were used to probe the TRAIL pathway. In addition to known genes, siRNAs targeting CDK4, PTGS1, ALG2, CLCN3, IRAK4, and MAP3K8 altered TRAIL-induced caspase-3 activation responses. Introduction of the miRNAs let-7c, mir-10a, mir-144, mir-150, mir-155, and mir-193 also affected the activation of the caspase cascade. Putative targets of these endogenous miRNAs included genes encoding death receptors, caspases, and other apoptosis-related genes. Among the novel genes revealed in the screen, CDK4 was selected for further characterization. CDK4 was the only member of the cyclin-dependent kinase gene family that bore a unique function in apoptotic signal transduction.

The let-7 microRNA represses cell proliferation pathways in human cells.

MicroRNAs play important roles in animal development, cell differentiation, and metabolism and have been implicated in human cancer. The let-7 microRNA controls the timing of cell cycle exit and terminal differentiation in Caenorhabditis elegans and is poorly expressed or deleted in human lung tumors. Here, we show that let-7 is highly expressed in normal lung tissue, and that inhibiting let-7 function leads to increased cell division in A549 lung cancer cells. Overexpression of let-7 in cancer cell lines alters cell cycle progression and reduces cell division, providing evidence that let-7 functions as a tumor suppressor in lung cells. let-7 was previously shown to regulate the expression of the RAS lung cancer oncogenes, and our work now shows that multiple genes involved in cell cycle and cell division functions are also directly or indirectly repressed by let-7. This work reveals the let-7 microRNA to be a master regulator of cell proliferation pathways.

Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification.

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3- to 5-fold by siRNA duplexes with 19 bp and 2 nt 3'-overhangs, whereas the maximum activation observed for poly(I)*poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N2-benzyl-2'-deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.

High-throughput RNAi screening in vitro: from cell lines to primary cells.

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.