MiR-34a Link to PD-L1 May Mean New Immunotherapy in NSCLC

Special Reports, NSCLC (Issue 7), Volume 7, Issue 2

Two presentations at this year’s American Association for Cancer Research (AACR) annual meeting linked a specific microRNA (miRNA), miR-34a, to an active area in immunotherapy, programed cell death-1 (PD-1) protein and its ligand, PD-L1.

James W. Welsh, MD

Two presentations at this year’s American Association for Cancer Research (AACR) annual meeting linked a specific microRNA (miRNA), miR-34a, to an active area in immunotherapy, the programed cell death-1 (PD-1) protein and its ligand, PD-L1.

The presentations1,2made by two colleagues at the University of Texas MD Anderson Cancer Center in Houston, James W. Welsh, MD, associate professor, Department of Radiation Oncology, Division of Radiation Oncology, and David S Hong, MD, deputy chair and associate professor, Department of Investigational Therapeutics, Division of Cancer Medicine. Together, the presentations explored the mechanism of action and safety of an agent mimicking miR-34a, MRX34, using preclinical and clinical data.3,4

Discovery and General Functions of MicroRNAs

In 1993 Lee et al discovered microRNAs in a widely used animal model.5Their work was praised for both its scientific perseverance and insight and was followed by the discovery of miRNAs in human tissues, including brain, heart, and lung, and across several other animal phyla.6,7

MiRNAs are short and noncoding. They post-transcriptionally modulate gene expression and therefore can regulate many gene products and cellular pathways.8There are more than 2000 identified miRNAs, variously classified as oncogenic, tumor suppressor, diagnostic, and prognostic.8,9Indeed, it has been found that there is a general down regulation of mature miRNAs in tumors versus normal tissues, and this has been taken to indicate the importance of constant, secure control of miRNA biosynthesis to ensure normal cellular physiology and development.8,10

The miR-34 Family, miR-34a and NSCLC

The miR-34 family includes miR-34a, miR-34b, and miR-34c. Among their targets are known oncogenes,c-MYC,MET,BCL-2,SIRT1,HDAC,SNAIL1,FOXP1, andCTNNB1, and they are principally involved in regulation of cell cycle progression and apoptosis.4,8,11,12Under conditions of no expression of miR-34, there is an increase in proliferation and survival via select target genes.8

MiR-34a has been shown to be transcriptionally induced by p53 and expressed at low levels in some cancers.12In a study of 70 patients with resected NSCLC and no therapy until relapse, low levels of miR-34a expression were correlated with a high probability of relapse.13Studies adding or administering miR-34a to cell and animal models of diffuse large B-cell lymphoma and lung cancer have indicated a possible therapeutic role for miR-34a. The growth of lung cancer xenografts in mice was blocked in response to systemically delivered synthetic miR-34a.14-16

miR-34a and PD-L1

Chen et al had previously demonstrated that miR-200 inhibited PD-L1, explaining how it prevented epithelial-to-mesenchymal transition and metastasis in lung cancer.17

Welsh et al have investigated the role of miR-34a in regulating PD-L1 activity. “We have worked with microRNAs in the past17,18 and shown that they can regulate many of the important pathways in cancer, so we turned our attention to how microRNAs can help a cancer cell evade the immune system and found the link between microRNA-34a and PD-L1,” said Welsh.

For their latest research, Welsh et al used specialized cell lines, NSCLC tissue samples from patients, and a syngeneic mouse model to answer specific research questions (Table).1

Table: Questions and the Techniques Used to Investigate the Role of miR-34a

Is PD-L1 a target of miR-34a?

Does p53 regulate PD-L1 via miR-34a?

What is the relationship between p53, PD-L1, and miR-34a in patients with NSCLC tumors?

What is the impact of miR-34a therapy plus radiation on PD-L1 expression and TILs?

Western blotting, Luciferase assays

In vitro models: p53-/-, p53+/+ HCT116 cells, p53-inducible H1299 cells, p53-knockdown

Determination of expression in formalin-fixed paraffin-embedded specimens

P53R172H∆g/+K-rasLA1/+ syngeneic mouse model with delivery of miR-34a-loaded liposomes (MRX34) with radiotherapy

PD-L1 indicates programed cell death-1 ligand; TIL, tumor infiltrating lymphocyte

The researchers reported that p53 does indeed regulate PD-L1 via miR-34a, which directly binds to the PD-L1 3’ untranslated region in NSCLC, repressing PD-L1. The delivery of miR-34a in the form of MRX34 to the syngeneic mouse model resulted in increased levels of CD8+ TILs and reduced levels of CD8+ PD-1+ cells. This effect was greater when radiation therapy was added and led to increased CD8+ cell numbers versus either therapy alone. There was a further benefit in that miR-34a delivery led to a reduction in macrophages and T-regulatory cells induced by the radiation therapy. This result leads to the possibility that a combination of miR-34a with standard therapies such as radiotherapy could be a new therapeutic option for patients with lung cancer.1

One challenge is identifying the patients for whom such a therapy would provide benefit. “Which patients [will] respond to anti-PD-1 therapy is still not known; however, our work supports a rationale that some types of p53 mutations have higher PD-L1 levels, [and] as such they may indeed have a better response to these types of drugs. While the rationale for p53 mutations is high, it [miR-34a] also regulates many other cancer pathways and may likely also benefit cancer patients with normal p53.”

In the Clinic

A phase I, open-label, multicenter, dose-escalation study of MRX34 started in April 2013 and is due to report in December 2015 (NCT01829971). The study enrolled patients with unresectable primary liver cancer or advanced metastatic cancer with liver involvement or hematologic malignancies. The primary endpoint is the maximum tolerated dose and the recommended phase II dose. The secondary endpoints are pharmacokinetics of MRX34 and the number of patients with evidence of clinical activity.

In a press release, Mirna Therapeutics described an interim analysis that was to be presented at this year’s AACR meeting by Hong.4The release stated that the safety profile was manageable, and that “molecular analysis of white blood cells from patients treated with MRX34 in the study shows a dose-dependent repression of several key oncogenes that have previously been identified as direct miR-34 targets, includingFOXP1,BCL2,HDAC1, andCTNNB1.” The press release concluded that this was evidence that miR-34 was entering human white blood cells and interacting with cellular targets of miRNA.

Commenting on what he would like to be the next steps, Welsh concluded, “We need to run the phase II clinical trials testing this approach for lung cancer, and in the lab we are looking for funding to test this therapy in other NSCLC cell lines and in combination with other cancer therapies like radiation.”

References

1. Cortez MA, Valdecanas D, Wang X, et al. p53 regulation of PD-L1 is mediated through miR-34a. American Association for Cancer Research Annual Meeting; April 18-22, Abstract 2875.

2. Hong D. Preclinical and clinical development of microRNA-34 mimi,cMRX34 for treatment of liver cancer. American Association for Cancer Research Annual Meeting; April 18-22, 2015.

3. MD Anderson Cancer Center Newsroom. MD Anderson study points to potential new lung cancer therapy http://www.mdanderson.org/newsroom/news-releases/2015/md-anderson-study-new-lung-cancer-therapy.html. Accessed May 1, 2015.

4. http://www.mirnarx.com/pdfs/releases/2015%200421%20Mirna%20AACR%20MRC34%20Interim%20Ph1.pdf. Accessed May 8, 2015.

5. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell.1993;75:843-854.

6. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004;116:281-297.

7. Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.Nature. 2000;408:86-89.

8. Barger JF, Nana-Sinkam SP. MicroRNA as tools and therapeutics in lung cancer [published online ahead of print February 19, 2015].Respir Med. 2015 Feb 19. doi:10.1016/j.rmed.2015.02.006.

9. Misso G, Di Martino MT, De Rosa G, et al. Mir-34: a new weapon against cancer?Mol Ther Nucleic Acids. 2014;3:e194.

10. Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers.Nature. 2005;435:834-838.

11. Wiggins JF, Ruffino L, Kelnar K, et al. Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34.Cancer Res. 2010;70:5923-5930.

12. Bommer GT, Gerin I, Feng Y, et al. p53-mediated activation of miRNA34 candidate tumor-suppressor genes.Curr Biol. 2007;17:1298-1307.

13. Gallardo E, Navarro A, Vinolas N, et al. miR-34a as a prognostic marker of relapse in surgically resected non-small-cell lung cancer.Carcinogenesis. 2009;30:1903-1909.

14. Craig VJ, Cogliatti SB, Imig J, et al. Myc-mediated repression of microRNA-34a promotes high-grade transformation of B-cell lymphoma by dysregulation of FoxP1.Blood. 2011;117:6227-6236.

15. Craig VJ, Tzankov A, Flori M, et al. Systemic microRNA-34a delivery induces apoptosis and abrogates growth of diffuse large B-cell lymphoma in vivo.Leukemia. 2012;26:2421-2424.

16. Wiggins JF, Ruffino L, Kelnar K, et al. Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34.Cancer Res. 2010;70:5923-5930.

17. Chen L, Gibbons DL, Goswami S, et al. Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression.Nat Commun. 2014;5:5241.

18. Cortez MA, Valdecanas D, Zhang X, et al. Therapeutic delivery of miR-200c enhances radiosensitivity in lung cancer.Mol Ther. 2014;22:1494-1503.

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