Heidi J. Peltier

Normalization of microRNA expression levels in quantitative RT-PCR assays: Identification of suitable reference RNA targets in normal and cancerous human solid tissues

Proper normalization is a critical but often an underappreciated aspect of quantitative gene expression analysis. This study describes the identification and characterization of appropriate reference RNA targets for the normalization of microRNA (miRNA) quantitative RT-PCR data. miRNA microarray data from dozens of normal and disease human tissues revealed ubiquitous and stably expressed normalization candidates for evaluation by qRT-PCR. miR-191 and miR-103, among others, were found to be highly consistent in their expression across 13 normal tissues and five pair of distinct tumor/normal adjacent tissues. These miRNAs were statistically superior to the most commonly used reference RNAs used in miRNA qRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA. The most stable normalizers were also highly conserved across flash-frozen and formalin-fixed paraffin-embedded lung cancer tumor/NAT sample sets, resulting in the confirmation of one well-documented oncomir (let-7a), as well as the identification of novel oncomirs. These findings constitute the first report describing the rigorous normalization of miRNA qRT-PCR data and have important implications for proper experimental design and accurate data interpretation.

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 p53R172HΔg/+K-rasLA1/+ 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.

Therapeutic Delivery of miR-200c Enhances Radiosensitivity in Lung Cancer

The microRNA (miR)-200s and their negative regulator ZEB1 have been extensively studied in the context of the epithelial-mesenchymal transition. Loss of miR-200s has been shown to enhance cancer aggressiveness and metastasis, whereas replacement of miR-200 miRNAs has been shown to inhibit cell growth in several types of tumors, including lung cancer. Here, we reveal a novel function of miR-200c, a member of the miR-200 family, in regulating intracellular reactive oxygen species signaling and explore a potential application for its use in combination with therapies known to increase oxidative stress such as radiation. We found that miR-200c overexpression increased cellular radiosensitivity by direct regulation of the oxidative stress response genes PRDX2, GAPB/Nrf2, and SESN1 in ways that inhibits DNA double-strand breaks repair, increase levels of reactive oxygen species, and upregulate p21. We used a lung cancer xenograft model to further demonstrate the therapeutic potential of systemic delivery of miR-200c to enhance radiosensitivity in lung cancer. Our findings suggest that the antitumor effects of miR-200c result partially from its regulation of the oxidative stress response; they further suggest that miR-200c, in combination with radiation, could represent a therapeutic strategy in the future.

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 × 101 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.

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy

Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development.

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.

Down-regulation of target gene expression in human white blood cells (hWBCs) by MRX34, a liposomal miR-34 mimic: Next generation sequencing (NGS) results from a first-in-human trial of microRNA cancer therapy.

e14078Background: Each microRNA (miRNA) modulates the expression of hundreds of genes across distinct cellular pathways, giving miRNA-based therapy the potential to simultaneously repress multiple ...

Abstract 4814: miRNA combination therapy: In vitro anticancer synergy between miR-34a mimic and next generation EGFR tyrosine kinase inhibitors (TKIs) in NSCLC

Background: miRNAs play a critical role in regulating key biological processes by modulating the expression of up to several hundred genes across multiple cellular pathways. miR-34a, one of the most widely studied miRNAs, is lost or expressed at reduced levels in many tumors, and normally functions as a natural tumor suppressor by down-regulating expression of >30 different oncogenes, as well as genes involved in tumor immune evasion, including PD-L1. MRX34 is a potential first-in-class liposome-encapsulated miR-34a mimic in Phase 1 study (NCT01829971) as monotherapy in patients with advanced malignancies. The ability of miR-34a to regulate the expression of key oncogenes across multiple oncogenic pathways makes MRX34 a rational candidate to combine with other anticancer therapies which are frequently subject to primary and acquired resistance in the clinic. Previous studies showed that miR-34a greatly sensitizes both EGFR wild-type and mutant NSCLC cell lines, as well as hepatocellular carcinoma cell lines, to the first generation EGFR TKI erlotinib. Here we report research combining miR-34a and the next generation EGFR TKIs afatinib (Gilotrif®) and rociletinib (CO-1686) in NSCLC cell lines. Methods: Combination studies using single-drug ratios (∼IC50 ratio of miR-34a and TKI) and multiple ratios above and below were performed in a panel of EGFR wild-type (A549, H460, H1299, H226) and EGFR mutant (H1975, HCC827 parent and HCC827 erl res) NSCLC cell lines. Cells were transfected with miR-34a and incubated 24 hrs later with afatinib or rociletinib for 72 hrs, with cellular proliferation then determined by AlamarBlue. Synergistic, additive, or antagonistic effects were determined by combination index (CI) values (based on Loewe9s concept of additivity), isobolograms, and curve-shift analyses. Results: Strong synergy was observed between miR-34a and both TKIs in all EGFR-mutant cell lines tested (CI Conclusions: Complementing previous results with miR-34a + erlotinib, the data demonstrate strongly synergistic anticancer effects between miR-34a and next generation EGFR TKIs in combination against a range of EGFR wild-type and mutant NSCLC cell lines. The results support clinical study of MRX34 + EGFR TKI combinations in patients with advanced NSCLC, including those with EGFR-mutant NSCLC that has progressed on EGFR TKI monotherapy. Citation Format: Jane Zhao, Adriana Guerrero, Kevin Kelnar, Heidi J. Peltier, Andreas G. Bader. miRNA combination therapy: In vitro anticancer synergy between miR-34a mimic and next generation EGFR tyrosine kinase inhibitors (TKIs) in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4814.

Abstract 2875: p53 regulation of PDL1 is mediated through miR-34a

Background Although clinical studies have shown promise for targeting PD1/PDL1 signaling in non-small cell lung cancer (NSCLC), little is known of how PDL1 expression is regulated. We previously found that miR-200s directly regulate PDL1; here we show that PDL1 is regulated by miR-34a and p53. Methods We confirmed that PDL1 is a direct target of miR-34a with western blotting and luciferase assays. We then used in vitro models (p53-/- and p53+/+ HCT116 cells, p53-inducible H1299 cells, and p53-knockdown H460 cells) to determine if p53 regulates PDL1 via miR-34a. Next, we analyzed p53, PDL1, and miR-34a expression in formalin-fixed paraffin-embedded specimens from patients with NSCLC. Finally, we used a p53R172HΔg/+K-rasLA1/+ syngeneic mouse model to deliver miR-34a-loaded liposomes (MRX34) plus radiotherapy and assessed PDL1 expression and tumor-infiltrating lymphocytes (TILs). Results We found that p53 regulates PDL1 via miR-34a, which directly binds to the PDL1 3′ untranslated region, in NSCLC. Delivery of MRX34 (previously tested in a phase I clinical trial) promoted CD8+ TILs and reduced CD8+PD1+ cells in vivo. Further, MRX34 plus radiotherapy increased CD8+ cell numbers more than either therapy alone. Finally, we showed that miR-34a delivery reduced the numbers of radiation-induced macrophages and T regulatory cells. Conclusions We identified a novel mechanism by which tumor immune evasion is regulated by p53 and miR-34a via PDL1. Our results suggest that delivery of miR-34a combined with standard therapies, such as radiotherapy, may represent a novel therapeutic approach for lung cancer. Citation Format: Maria A. Cortez, David Valdecanas, Xiaohong Wang, Cristina Ivan, Heidi Peltier, Huiping Ye, Luiz Araujo, David Carbone, Dipak K. Giri, Ritsuko Komaki, Sunil Krishnan, Ferdinandos Skoulidis, John Heymach, George Calin, Andreas G. Bader, James W. Welsh. p53 regulation of PDL1 is mediated through miR-34a. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2875. doi:10.1158/1538-7445.AM2015-2875