Jane Zhao

A combinatorial microRNA therapeutics approach to suppressing non-small cell lung cancer

Targeted cancer therapies, although often effective, have limited utility owing to preexisting primary or acquired secondary resistance. Consequently, agents are sometimes used in combination to simultaneously affect multiple targets. MicroRNA mimics are excellent therapeutic candidates because of their ability to repress multiple oncogenic pathways at once. Here we treated the aggressive Kras;p53 non-small cell lung cancer mouse model and demonstrated efficacy with a combination of two tumor-suppressive microRNAs (miRNAs). Systemic nanodelivery of miR-34 and let-7 suppressed tumor growth leading to survival advantage. This combinatorial miRNA therapeutic approach engages numerous components of tumor cell-addictive pathways and highlights the ability to deliver multiple miRNAs in a safe and effective manner to target lung tissue.

Systemic Delivery of a miR34a Mimic as a Potential Therapeutic for Liver Cancer

miR34a is a tumor-suppressor miRNA that functions within the p53 pathway to regulate cell-cycle progression and apoptosis. With apparent roles in metastasis and cancer stem cell development, miR34a provides an interesting opportunity for therapeutic development. A mimic of miR34a was complexed with an amphoteric liposomal formulation and tested in two different orthotopic models of liver cancer. Systemic dosing of the formulated miR34a mimic increased the levels of miR34a in tumors by approximately 1,000-fold and caused statistically significant decreases in the mRNA levels of several miR34a targets. The administration of the formulated miR34a mimic caused significant tumor growth inhibition in both models of liver cancer, and tumor regression was observed in more than one third of the animals. The antitumor activity was observed in the absence of any immunostimulatory effects or dose-limiting toxicities. Accumulation of the formulated miR34a mimic was also noted in the spleen, lung, and kidney, suggesting the potential for therapeutic use in other cancers. Mol Cancer Ther; 13(10); 2352–60. ©2014 AACR.

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.

TP53-independent Function of miR-34a via HDAC1 and p21CIP1/WAF1

The tumor suppressor, microRNA-34 (miR-34), a transcriptional target of TP53, functions in a positive feedback loop to activate TP53. Although miR-34 can inhibit cancer cells carrying TP53 mutations, this feedback to TP53 may be a prerequisite for full miR-34 function and may restrict its therapeutic application to patients with intact TP53. To investigate the functional relationships between TP53 and miR-34, and that of other TP53-regulated miRNAs including miR-215/192, we have used a panel of isogenic cancer cell lines that differ only with respect to their endogenous TP53 status. miR-34-induced inhibition of cancer cell growth is the same in TP53-positive and TP53-negative cells. In contrast, miR-215/192 functions through TP53. In the absence of TP53, miR-34, but not miR-215/192, is sufficient to induce an upregulation of the cell cycle-dependent kinase inhibitor p21(CIP1/WAF1). We identify histone deacetylase 1 (HDAC1) as a direct target of miR-34 and demonstrate that repression of HDAC1 leads to an induction of p21(CIP1/WAF1) and mimics the miR-34 cellular phenotype. Depletion of p21(CIP1/WAF1) specifically interferes with the ability of miR-34 to inhibit cancer cell proliferation. The data suggest that miR-34 controls a tumor suppressor pathway previously reserved for TP53 and provides an attractive therapeutic strategy for cancer patients irrespective of TP53 status.The tumor suppressor, microRNA-34 (miR-34), a transcriptional target of TP53, functions in a positive feedback loop to activate TP53. Although miR-34 can inhibit cancer cells carrying TP53 mutations, this feedback to TP53 may be a prerequisite for full miR-34 function and may restrict its therapeutic application to patients with intact TP53. To investigate the functional relationships between TP53 and miR-34, and that of other TP53-regulated miRNAs including miR-215/192, we have used a panel of isogenic cancer cell lines that differ only with respect to their endogenous TP53 status. miR-34-induced inhibition of cancer cell growth is the same in TP53-positive and TP53-negative cells. In contrast, miR-215/192 functions through TP53. In the absence of TP53, miR-34, but not miR-215/192, is sufficient to induce an upregulation of the cell cycle-dependent kinase inhibitor p21CIP1/WAF1. We identify histone deacetylase 1 (HDAC1) as a direct target of miR-34 and demonstrate that repression of HDAC1 leads to an induction of p21CIP1/WAF1 and mimics the miR-34 cellular phenotype. Depletion of p21CIP1/WAF1 specifically interferes with the ability of miR-34 to inhibit cancer cell proliferation. The data suggest that miR-34 controls a tumor suppressor pathway previously reserved for TP53 and provides an attractive therapeutic strategy for cancer patients irrespective of TP53 status.

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.

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 4829: miRNA combination therapy: In vitro anticancer synergy between miR-34a mimic and cytotoxic chemotherapy (CT) 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 EGFR tyrosine kinase inhibitor erlotinib. Here we report research combining miR-34a and standard CT drugs in NSCLC cell lines. Methods: Combination studies using single-drug ratios (∼IC50 ratio of miR-34a and CT drug) and multiple ratios above and below were performed in a panel of NSCLC cell lines (A549, H460, H1299, H2073) with varying degrees of intrinsic resistance to CT. Cells were transfected with miR-34a and incubated 24 hrs later with cisplatin, carboplatin, paclitaxel, gemcitabine, or pemetrexed 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: Synergistic interactions were observed between miR-34 and all CT drugs in all NSCLC cell lines tested. Synergy was observed at multiple miR-34a/cytotoxic agent dose ratios and at drug concentrations inducing 50% or greater inhibition (CI 2 at both 50% and 75% effect levels). Stronger synergy was seen in the H2073 cell line which is relatively more resistant to CT. Conclusions: Consistent with other in vitro results with miR-34a-based combinations, the data demonstrate synergistic anticancer effects between miR-34a + CT in a range of NSCLC cell lines with varying degrees of intrinsic resistance to CT. Overall, the data support further exploration of MRX34 in combination with standard anticancer therapies, and we are evaluating the most appropriate combinations for near-term clinical trials. In addition to resistance, other critical oncogenic mechanisms present in the tumor microenvironment (eg, immune evasion, cancer stem cells, metastasis) could potentially also be mitigated to the benefit of patients by a miRNA-based approach to therapy. Citation Format: Adriana Guerrero, Jane Zhao, Xiaojie Yu, Alexander Pertsemlidis, Andreas G. Bader. miRNA combination therapy: In vitro anticancer synergy between miR-34a mimic and cytotoxic chemotherapy (CT) 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 4829.

Abstract B57: miR-34 mimics synergize with small molecule inhibitors targeting the EGFR and Raf kinase pathways

Sorafenib (Nexavar®) and erlotinib (Tarceva®) are FDA-approved therapies for patients with hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC), respectively. Sorafenib is a multi-kinase inhibitor targeting the Raf/Mek/Erk pathway, and erlotinib is a tyrosine kinase inhibitor antagonizing epidermal growth factor receptor (EGFR). Due to their selective inhibitory action and observed toxicities, current treatment options for liver and lung cancer are limited and many cancers develop resistance. Naturally occurring tumor suppressor microRNAs inhibit tumor growth by regulating multiple oncogenes at once and, therefore, microRNA mimics, which are copies of the naturally occurring microRNAs, may be used in combination with the respective standard of care drugs to bring this tumor suppressor activity back into tumor cells and thereby augment drug sensitivity. Here, we investigated the relationship of a mimic of the tumor suppressor microRNA miR-34 in combination with erlotinib or sorafenib and determined the therapeutic activity of the combination in lung and liver cancer cells. Data derived from isobolograms, combination index plots and curve shift analyses indicate synergy in all cancer cell lines tested. Synergy was observed at multiple microRNA and drug ratios and at drug concentrations that induce 50% or greater cancer cell inhibition. Data from cell and animal studies will be presented. Citation Format: Jane Zhao, Kevin Kelnar, Chris Daige, Jason Wiggins, Leslie Priddy, Terri Muenzer, Julie Tran, David Brown, Andreas G. Bader. miR-34 mimics synergize with small molecule inhibitors targeting the EGFR and Raf kinase pathways. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B57.

Abstract LB-250: Development of a miR34-based cancer therapy.

miR-34 is a tumor suppressor miRNA that functions within the p53 pathway and regulates the expression of more than twenty oncogenes, including MET, MYC, WNT, and MYB. We have created a mimic of miR-34 called miR-Rx34 that induces cell cycle arrest, senescence, and apoptosis in cancer cells. Transient transfection of miR-Rx34 significantly reduces the proliferation of cancer cells derived from patients with liver, lung, breast, colon, and pancreatic cancers as well as melanoma, lymphoma, and multiple myeloma. miR-Rx34 has been encapsulated in an anionic liposome to produce a particle called MRX34 that has a diameter of ~120 nm. Intravenous injections of MRX34 causes dose-dependent increases in miR-34 levels in liver tumor, lung tumor, liver, lung, spleen, and other highly vascularized tissues in mice without inducing an immune response. Systemic delivery of MRX34 causes complete tumor regression in two orthotopic models of liver cancer. The exceptional potency of MRX34 likely results from its capacity to stimulate the p53 pathway and down-regulate genes in the wnt/B-catenin, MET, MAPK, and VegF pathways in liver tumors. The no adverse effects level observed for MRX34 in IND-enabling toxicology studies in monkeys and rats are 10-100-fold higher than the doses that produce complete tumor regression in mice, suggesting that the drug candidate will have a broad therapeutic window in cancer patients. Clinical studies with MRX34 that are expected to begin in early 2013 will provide the first measure of the therapeutic potential of a tumor suppressor miRNA. Citation Format: Chris Daige, Jason Wiggins, Leslie Priddy, Kevin Kelnar, Jane Zhao, Andreas Bader, David Brown. Development of a miR34-based cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-250. doi:10.1158/1538-7445.AM2013-LB-250

Abstract 5636: Preclinical data of a microRNA-based therapy for hepatocellular carcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL MicroRNA (miRNA) mimics have emerged as a novel class of therapeutics with promising anti-oncogenic activity. These mimics are modeled after naturally occurring tumor suppressor miRNAs that are ubiquitously expressed in normal cells but frequently show a loss-of-function in human malignancies. The premise for the strong inhibitory activity is based on the observation these endogenous miRNAs control multiple oncogenic pathways commonly deregulated in cancer. Therefore, “miRNA replacement therapy” acts in accordance with our current understanding of cancer as a pathway disease that can only be successfully treated when intervening with multiple cancer pathways. We have identified a series of key tumor suppressor miRNAs, including miR-34, and validated the therapeutic potential in cultured cancer cells and mouse models of cancer. The translation of this potential into future medicines, however, was hampered by the lack of a robust clinically relevant delivery system. To facilitate a rapid route to the clinic, we have screened a panel of external delivery systems that are in pre-clinical development or have already reached the clinic featuring another oligonucleotide. Here, we present the pharmacologic and pharmacodynamic parameters of miRNA mimics complexed in ionizable NOV340 liposomes (SMARTICLEs, Marina Biotech, Bothell, WA) in an orthotopic tumor model of hepatocellular carcinoma. Treatment of mice carrying existing tumors mimics led to significant tumor regression, prolonged survival and lacked notable drug-related side effects. Some of the mice appeared to be tumor-free. The data demonstrate the therapeutic utility of the NOV340/miRNA formulation and support the initiation of IND-enabling studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5636. doi:1538-7445.AM2012-5636