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Dual Inhibitors-Loaded Nanotherapeutics that Target Kinase Signaling Pathways Synergize with Immune Checkpoint Inhibitor

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Abstract

Introduction

Immune checkpoint inhibitors that boost cytotoxic T cell-based immune responses have emerged as one of the most promising approaches in cancer treatment. However, it is increasingly being realized that T cell activation needs to be rationally combined with molecularly targeted therapeutics for a maximal anti-tumor outcome. Currently, two oncogenic drivers, MAPK and PI3K-mTOR have emerged as the two main molecular targets for combining with immunotherapy. However, there are major challenges in enabling such combinations: first, such combinations can result in high rates of toxicity. Second, while, these molecular targets could be driving tumor progression, they are essential for activation of the immune cells. So, the kinase inhibitors and immunotherapy can antagonize each other.

Objectives

We rationalized that the synergistic combination of kinase inhibitors and immunotherapy could be enabled by dual inhibitors-loaded supramolecular nanotherapeutics (DiLN) that can co-deliver PI3K- and MAPK-inhibitors to the cancer cells and activate immune response by T cell-modulating immunotherapy, resulting in greater anti-tumor efficacy while minimizing toxicity.

Methods

We engineered DiLNs by designing the amphiphilic building blocks (both drugs and co-lipids) that enables supramolecular nanoassembly. DiLNs were tested for their physiochemical properties including size, morphology, stability and drug release kinetics profiles. The efficacy of DiLNs was tested in drug-resistant cells such as BRAFV600E melanoma (D4M), Clear cell ovarian carcinoma (TOV21G) cells. The tumor inhibition efficiency of DiLNs in combination with immune checkpoint inhibitor antibody was studied in syngeneic D4M animal model.

Results

DiLNs were stable for over a month and released the drugs in a sustained manner. In vitro cytotoxicity studies in D4M and TOV21G cells showed that DiLNs were significantly more effective than free drugs. In vivo studies showed that the combination of DiLNs with anti PD-L1 antibody resulted in superior antitumor effect and survival.

Conclusion

This study shows that the rational combination of DiLNs that target multiple oncogenic signaling pathways with immune checkpoint inhibitors could emerge as an effective strategy to improve immunotherapeutic response against drug resistant tumors.

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Abbreviations

PI3K:

Phosphoinositide 3-kinase

mTOR:

Mammalian target of rapamycin

MAPK:

Mitogen-activated protein kinase kinase

SNP:

Supramolecular nanoparticles/nanotherapeutics

EGFR:

Epidermal growth factor receptor

D4M:

Dartmouth murine mutant malignant melanoma-3A

Akt:

Protein kinase B

RTK:

Receptor tyrosine kinase

PD-L1:

Programed death ligand 1

MAPK:

Mitogen-activated protein kinases

Erk:

Extracellular signal-regulated kinase

mAb:

Monoclonal antibody

FDA:

Food and drug administration

PD-1:

Programmed death protein 1

CTLA-4:

Cytotoxic T-lymphocyte-associated protein 4

OCCC:

Ovarian clear cell carcinoma

PBMCs:

Peripheral blood monomorphonuclear cells

PBS:

Phosphate buffered saline

TUNEL:

Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling

NiR:

Near infra-red

OCT:

Optimal cutting temperature

References

  1. Bekaii-Saab, T., et al. Multi-institutional phase II study of selumetinib in patients with metastatic biliary cancers. J. Clin. Oncol. 29:2357–2363, 2011. https://doi.org/10.1200/JCO.2010.33.9473.

    Article  Google Scholar 

  2. Brahmer, J. R., et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366:2455–2465, 2012. https://doi.org/10.1056/NEJMoa1200694.

    Article  Google Scholar 

  3. Burotto, M., V. L. Chiou, J. M. Lee, and E. C. Kohn. The MAPK pathway across different malignancies: a new perspective. Cancer 120:3446–3456, 2014. https://doi.org/10.1002/cncr.28864.

    Article  Google Scholar 

  4. Cho, K., X. Wang, S. Nie, Z. G. Chen, and D. M. Shin. Therapeutic nanoparticles for drug delivery in cancer. Clin. Cancer Res 14:1310–1316, 2008. https://doi.org/10.1158/1078-0432.CCR-07-1441.

    Article  Google Scholar 

  5. Choi, K. C., N. Auersperg, and P. C. Leung. Mitogen-activated protein kinases in normal and (pre)neoplastic ovarian surface epithelium. Reprod Biol Endocrinol 1:71, 2003. https://doi.org/10.1186/1477-7827-1-71.

    Article  Google Scholar 

  6. Coates, A., et al. On the receiving end–patient perception of the side-effects of cancer chemotherapy. Eur. J. Cancer Clin. Oncol. 19:203–208, 1983.

    Article  Google Scholar 

  7. Davis, M. E., Z. G. Chen, and D. M. Shin. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat. Rev. Drug Discov. 7:771–782, 2008. https://doi.org/10.1038/nrd2614.

    Article  Google Scholar 

  8. Deken, M. A., et al. Targeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma. Oncoimmunology 5:e1238557, 2016. https://doi.org/10.1080/2162402X.2016.1238557.

    Article  Google Scholar 

  9. Dhillon, A. S., S. Hagan, O. Rath, and W. Kolch. MAP kinase signalling pathways in cancer. Oncogene 26:3279, 2007. https://doi.org/10.1038/sj.onc.1210421.

    Article  Google Scholar 

  10. Dienstmann, R., J. Rodon, V. Serra, and J. Tabernero. Picking the point of inhibition: a comparative review of PI3K/AKT/mTOR pathway inhibitors. Mol Cancer Ther. 13:1021–1031, 2014. https://doi.org/10.1158/1535-7163.MCT-13-0639.

    Article  Google Scholar 

  11. Eliopoulos, A. G., et al. The control of apoptosis and drug resistance in ovarian cancer: influence of p53 and Bcl-2. Oncogene 11:1217–1228, 1995.

    Google Scholar 

  12. Fruman, D. A., et al. The PI3K pathway in human disease. Cell 170:605–635, 2017. https://doi.org/10.1016/j.cell.2017.07.029.

    Article  Google Scholar 

  13. Goldman, A., et al. Rationally designed 2-in-1 nanoparticles can overcome adaptive resistance in cancer. ACS Nano 10:5823–5834, 2016. https://doi.org/10.1021/acsnano.6b00320.

    Article  Google Scholar 

  14. Holderfield, M., M. M. Deuker, F. McCormick, and M. McMahon. Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond. Nat. Rev. Cancer 14:455–467, 2014. https://doi.org/10.1038/nrc3760.

    Article  Google Scholar 

  15. Holohan, C., S. Van Schaeybroeck, D. B. Longley, and P. G. Johnston. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer 13:714–726, 2013. https://doi.org/10.1038/nrc3599.

    Article  Google Scholar 

  16. Hong, D. S., et al. A multicenter phase I trial of PX-866, an oral irreversible phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors. Clin. Cancer Res. 18:4173–4182, 2012. https://doi.org/10.1158/1078-0432.CCR-12-0714.

    Article  Google Scholar 

  17. Housman, G., et al. Drug resistance in cancer: an overview. Cancers (Basel) 6:1769–1792, 2014. https://doi.org/10.3390/cancers6031769.

    Article  Google Scholar 

  18. Inamdar, G. S., S. V. Madhunapantula, and G. P. Robertson. Targeting the MAPK pathway in melanoma: why some approaches succeed and other fail. Biochem. Pharmacol. 80:624–637, 2010. https://doi.org/10.1016/j.bcp.2010.04.029.

    Article  Google Scholar 

  19. Janku, F., T. A. Yap, and F. Meric-Bernstam. Targeting the PI3K pathway in cancer: are we making headway? Nat. Rev. Clin. Oncol. 15:273–291, 2018. https://doi.org/10.1038/nrclinonc.2018.28.

    Article  Google Scholar 

  20. Kim, E. H., and M. Suresh. Role of PI3K/Akt signaling in memory CD8 T cell differentiation. Front. Immunol. 4:20, 2013. https://doi.org/10.3389/fimmu.2013.00020.

    Article  Google Scholar 

  21. Kirkwood, J. M., et al. Next generation of immunotherapy for melanoma. J. Clin. Oncol. 26:3445–3455, 2008. https://doi.org/10.1200/JCO.2007.14.6423.

    Article  Google Scholar 

  22. Kulkarni, A., S. K. Natarajan, V. Chandrasekar, P. R. Pandey, and S. Sengupta. Combining immune checkpoint inhibitors and kinase-inhibiting supramolecular therapeutics for enhanced anticancer efficacy. ACS Nano 10:9227–9242, 2016. https://doi.org/10.1021/acsnano.6b01600.

    Article  Google Scholar 

  23. Kulkarni, A. A., et al. Supramolecular nanoparticles that target phosphoinositide-3-kinase overcome insulin resistance and exert pronounced antitumor efficacy. Cancer Res 73:6987–6997, 2013. https://doi.org/10.1158/0008-5472.CAN-12-4477.

    Article  Google Scholar 

  24. Kulkarni, A., et al. A designer self-assembled supramolecule amplifies macrophage immune responses against aggressive cancer. Nat. Biomed. Eng. 2:589–599, 2018. https://doi.org/10.1038/s41551-018-0254-6.

    Article  Google Scholar 

  25. Kwong, L. N., and M. A. Davies. Navigating the therapeutic complexity of PI3K pathway inhibition in melanoma. Clin. Cancer Res. 19:5310–5319, 2013. https://doi.org/10.1158/1078-0432.CCR-13-0142.

    Article  Google Scholar 

  26. Lee, S., E. J. Choi, C. Jin, and D. H. Kim. Activation of PI3K/Akt pathway by PTEN reduction and PIK3CA mRNA amplification contributes to cisplatin resistance in an ovarian cancer cell line. Gynecol Oncol 97:26–34, 2005. https://doi.org/10.1016/j.ygyno.2004.11.051.

    Article  Google Scholar 

  27. Long, G. V., et al. Combined BRAF and MEK inhibition versus BRAF inhibition alone in melanoma. N. Engl. J. Med. 371:1877–1888, 2014. https://doi.org/10.1056/NEJMoa1406037.

    Article  Google Scholar 

  28. Mabuchi, S., T. Sugiyama, and T. Kimura. Clear cell carcinoma of the ovary: molecular insights and future therapeutic perspectives. J. Gynecol. Oncol. 27:e31, 2016. https://doi.org/10.3802/jgo.2016.27.e31.

    Article  Google Scholar 

  29. Mahoney, K. M., G. J. Freeman, and D. F. McDermott. The next immune-checkpoint inhibitors: PD-1/PD-L1 blockade in melanoma. Clin Ther 37:764–782, 2015. https://doi.org/10.1016/j.clinthera.2015.02.018.

    Article  Google Scholar 

  30. Manzano, J. L., et al. Resistant mechanisms to BRAF inhibitors in melanoma. Ann. Transl. Med. 4:237, 2016. https://doi.org/10.21037/atm.2016.06.07.

    Article  Google Scholar 

  31. Mayer, I. A., and C. L. Arteaga. The PI3K/AKT pathway as a target for cancer treatment. Annu Rev. Med. 67:11–28, 2016. https://doi.org/10.1146/annurev-med-062913-051343.

    Article  Google Scholar 

  32. Mendoza, M. C., E. E. Er, and J. Blenis. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem. Sci. 36:320–328, 2011. https://doi.org/10.1016/j.tibs.2011.03.006.

    Article  Google Scholar 

  33. Park, S., et al. PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, has antileukemic activity in AML. Leukemia 22:1698–1706, 2008. https://doi.org/10.1038/leu.2008.144.

    Article  Google Scholar 

  34. Peng, W., et al. Loss of PTEN promotes resistance to T cell-mediated immunotherapy. Cancer Discov 6:202–216, 2016. https://doi.org/10.1158/2159-8290.CD-15-0283.

    Article  Google Scholar 

  35. Pino, P. D., et al. Protein corona formation around nanoparticles—from the past to the future. Mater. Horiz. 1:301–313, 2014. https://doi.org/10.1039/c3mh00106g.

    Article  Google Scholar 

  36. Pons-Tostivint, E., B. Thibault, and J. Guillermet-Guibert. Targeting PI3K signaling in combination cancer therapy. Trends Cancer 3:454–469, 2017. https://doi.org/10.1016/j.trecan.2017.04.002.

    Article  Google Scholar 

  37. Prieto, P. A., et al. CTLA-4 blockade with ipilimumab: long-term follow-up of 177 patients with metastatic melanoma. Clin. Cancer Res. 18:2039–2047, 2012. https://doi.org/10.1158/1078-0432.CCR-11-1823.

    Article  Google Scholar 

  38. Raynaud, F. I., et al. Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941. Mol. Cancer Ther. 8:1725–1738, 2009. https://doi.org/10.1158/1535-7163.MCT-08-1200.

    Article  Google Scholar 

  39. Santarpia, L., S. M. Lippman, and A. K. El-Naggar. Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy. Expert Opin. Ther. Targets 16:103–119, 2012. https://doi.org/10.1517/14728222.2011.645805.

    Article  Google Scholar 

  40. Sarker, D., et al. First-in-human phase I study of pictilisib (GDC-0941), a potent pan-class I phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with advanced solid tumors. Clin. Cancer Res. 21:77–86, 2015. https://doi.org/10.1158/1078-0432.CCR-14-0947.

    Article  Google Scholar 

  41. Shapiro, G. I., et al. Phase I safety, pharmacokinetic, and pharmacodynamic study of SAR245408 (XL147), an oral pan-class I PI3K inhibitor, in patients with advanced solid tumors. Clin. Cancer Res. 20:233–245, 2014. https://doi.org/10.1158/1078-0432.CCR-13-1777.

    Article  Google Scholar 

  42. Sharp, L. L., D. A. Schwarz, C. M. Bott, C. J. Marshall, and S. M. Hedrick. The influence of the MAPK pathway on T cell lineage commitment. Immunity 7:609–618, 1997.

    Article  Google Scholar 

  43. Siegel, R. L., K. D. Miller, and A. Jemal. Cancer statistics, 2018. CA Cancer. J. Clin. 68:7–30, 2018. https://doi.org/10.3322/caac.21442.

    Article  Google Scholar 

  44. Smalley, K. S., et al. Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases. Mol Cancer Ther 5:1136–1144, 2006. https://doi.org/10.1158/1535-7163.MCT-06-0084.

    Article  Google Scholar 

  45. Sordella, R., D. W. Bell, D. A. Haber, and J. Settleman. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 305:1163–1167, 2004. https://doi.org/10.1126/science.1101637.

    Article  Google Scholar 

  46. Topalian, S. L., C. G. Drake, and D. M. Pardoll. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell 27:450–461, 2015. https://doi.org/10.1016/j.ccell.2015.03.001.

    Article  Google Scholar 

  47. Vanneman, M., and G. Dranoff. Combining immunotherapy and targeted therapies in cancer treatment. Nat Rev. Cancer 12:237–251, 2012. https://doi.org/10.1038/nrc3237.

    Article  Google Scholar 

  48. Vara, J. A. F., et al. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev 30:193–204, 2004. https://doi.org/10.1016/j.ctrv.2003.07.007.

    Article  Google Scholar 

  49. Vella, L. J., et al. MEK inhibition, alone or in combination with BRAF inhibition, affects multiple functions of isolated normal human lymphocytes and dendritic cells. Cancer Immunol Res 2:351–360, 2014. https://doi.org/10.1158/2326-6066.CIR-13-0181.

    Article  Google Scholar 

  50. Villanueva, J., et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 18:683–695, 2010. https://doi.org/10.1016/j.ccr.2010.11.023.

    Article  Google Scholar 

  51. Wang, X., et al. Tumor suppressor miR-34a targets PD-L1 and functions as a potential immunotherapeutic target in acute myeloid leukemia. Cell. Signal. 27:443–452, 2015. https://doi.org/10.1016/j.cellsig.2014.12.003.

    Article  Google Scholar 

  52. Wilmott, J. S., et al. Selective BRAF inhibitors induce marked T-cell infiltration into human metastatic melanoma. Clin. Cancer Res. 18:1386–1394, 2012. https://doi.org/10.1158/1078-0432.CCR-11-2479.

    Article  Google Scholar 

  53. Xu, S., et al. miR-424(322) reverses chemoresistance via T-cell immune response activation by blocking the PD-L1 immune checkpoint. Nat Commun 7:11406, 2016. https://doi.org/10.1038/ncomms11406.

    Article  Google Scholar 

  54. Yamazaki, Y., et al. Difference between cancer cells and the corresponding normal tissue in view of stereoselective hydrolysis of synthetic esters. Biochim. Biophys. Acta 1243:300–308, 1995.

    Article  Google Scholar 

  55. Zhao, Y., and A. A. Adjei. The clinical development of MEK inhibitors. Nat. Rev. Clin. Oncol. 11:385–400, 2014. https://doi.org/10.1038/nrclinonc.2014.83.

    Article  Google Scholar 

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Acknowledgments

We are extremely grateful for the support offered by the Brigham & Women’s Hospital Young Investigator Award, Melanoma Research Alliance Young Investigator Award (510283) and Cancer Research Institute (118-1501) Technology Impact Award to A. K. We would like to thank the BWH animal facility for their help with in vivo imaging. We thank the Biophysical Characterization Core at the Institute for Applied Life Sciences (IALS), University of Massachusetts Amherst for lending their expertise in regards to characterization experiments. We would also like thank Light Microscopy Core facility at University of Massachusetts Amherst for their help and consultation while performing confocal imaging.

Conflict of interest

Anujan Ramesh, Siva Kumar Natarajan, Dipika Nandi and Ashish Kulkarni declare that they have no conflicts of interest.

Research Involving in Human and Animal Studies

All institutional and national guidelines for the care and use of laboratory animals were followed and approved by the appropriate institutional committees. No human studies were carried out by the authors for this article.

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Author notes

  1. Anujan Ramesh and Siva Kumar Natarajan contributed equally to this study.

Authors and Affiliations

  1. Department of Chemical Engineering, University of Massachusetts, Amherst, MA, USA

    Anujan Ramesh & Ashish Kulkarni

  2. Department of Veterinary and Animal Science, University of Massachusetts, Amherst, MA, USA

    Dipika Nandi & Ashish Kulkarni

  3. Center for Bioactive Delivery, Institute for Applied Life Sciences, University of Massachusetts, Amherst, MA, USA

    Ashish Kulkarni

  4. Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Siva Kumar Natarajan & Ashish Kulkarni

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  1. Anujan Ramesh
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Correspondence to Ashish Kulkarni.

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Ashish Kulkarni is an Assistant Professor in the Department of Chemical Engineering at the University of Massachusetts Amherst. Prior to this, he was an Instructor of Medicine at Harvard Medical School and Associate Bioengineer at Brigham & Women’s Hospital. He obtained his B. Tech. in Chemical Technology from Institute of Chemical Technology, University of Mumbai and a PhD in Chemistry from University of Cincinnati, Ohio. He completed his postdoctoral training with Prof. Shiladitya Sengupta at Harvard Medical School and MIT. In Prof. Sengupta’s laboratory, his research efforts were focused on the development of structure–activity relationship-inspired nanomedicine for cancer therapy. His lab is currently working on the development of tools and platform technologies for immunotherapy applications. His work has been published in Nature Biomedical Engineering, Nature Communications, PNAS, ACS Nano and Cancer Research, and featured in several science media outlets. He was recently selected as one of the top 12 rising researchers (‘Talented 12′) by American Chemical Society’s (ACS) Chemical & Engineering News and ‘NextGen Star’ in Cancer Research by American Association for Cancer Research (AACR). He is a recipient of several awards including American Cancer Society Research Scholar Award, Melanoma Research Alliance Young Investigator Award, Cancer Research Institute Technology Impact Award, Hearst Foundation Young Investigator Award, Harvard Cancer Center Career Development Award, AACR Scholar-in-training Award, American Society of Pharmacology and Experimental Therapeutics (ASPET) Young Scientist Award and Brigham & Women’s Hospital Junior Faculty Mentor Award.

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Ramesh, A., Natarajan, S.K., Nandi, D. et al. Dual Inhibitors-Loaded Nanotherapeutics that Target Kinase Signaling Pathways Synergize with Immune Checkpoint Inhibitor. Cel. Mol. Bioeng. 12, 357–373 (2019). https://doi.org/10.1007/s12195-019-00576-1

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