PTEN Mutations Trigger Resistance to Immunotherapy Feixiong Cheng1,2,3,* and Charis Eng1,2,3,4,5,* Immune checkpoint-blocking antibodies are actively used to treat multiple cancer types; however, the underlying resistance mechanism remains unclear. In a recent study, Zhao et al. (Nat. Med. 2019;25:462–469) found that somatic PTEN mutations were associated with resistance to immune checkpoint inhibitors by altering immunosuppressive environments in patients with glioblastomas. Immune checkpoint monoclonal antibody blockades, such as programed cell death 1 (PD-1) inhibitors, have led to remarkable and durable antitumor effects in multiple cancer types, including melanoma and non-small cell lung cancer . However, less than 10% of patients with glioblastomas (GBM) have long-term responses and the molecular determinants in the resistance mechanisms to this immunotherapy are poorly characterized. Zhao and colleagues performed a longitudinal study of 66 patients during standard therapy and after anti-PD-1 immunotherapies [nivolumab (trade name: Opdivo) or pembrolizumab (trade name: Keytruda)], including 17 long-term responders who showed durable responses to therapy . Via wholeexome sequencing analysis, they found that somatic mutations on phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were signiﬁcantly enriched in nonresponders after correcting the background mutation rate
derived from The Cancer Genome Atlas (TCGA). Speciﬁcally, they found 23 somatic mutations (including missense, frameshift indel, and nonsense mutations) in PTEN among the 32 nonresponders, but only three PTEN mutations among the 13 responders. For example, 3D protein structural analysis revealed multiple loss-of-function (LoF) mutations (Figure 1), including D107Y, G132D, K147*, and R173C in the phosphatase domain of PTEN, and R233*, D252Y, and L295fs within the C2 domain of PTEN. As a consequence these PTEN LoF mutations canonically activate the phosphatidylinositol 3kinase (PI3K) and AKT (PI3K-AKT) pathway . By individual sample gene-set enrichment analysis, Zhao and colleagues showed signiﬁcantly elevated PI3K-AKT pathway activity in nonresponsive tumors with PTEN somatic mutations .
involved in immune system responses) transcriptional signature , an observation corroborated by reduced CD4+FOXP3+ Treg cell numbers in subjects with germline PTEN mutations . Furthermore, correlation analysis showed that somatic PTEN mutations were signiﬁcantly associated with elevated levels of neutrophils, microglia, and macrophages in the tumor microenvironment. They further used quantitative multiplex immunoﬂuorescence to inspect the structure of the tumor microenvironment triggered by PTEN mutations. By analyzing formalin-ﬁxed parafﬁn-embedded specimens from 17 patients with matched pre- and post-anti-PD-1 treatment samples (including ten responders and seven nonresponders), they showed that PTEN-mutant tumors were more likely to have elevated levels of CD68+ (cluster of differentiation 68) macrophage inﬁltration. Furthermore, CD68+HLADR macrophages, a speciﬁc cell subpopulation that is associated with poor survival in melanoma, was signiﬁcantly elevated in PTEN-mutant tumors . Using spatial statistics-based pair correlation analysis, they demonstrated that the clustering of tumor cells with each other in PTEN-mutant tumors was significant when compared with PTEN wild type cases before immunotherapy. In addition, macrophages were signiﬁcantly clustered with each other in PTEN wild type tumors compared with PTENmutant cases after immunotherapy, indicating that PTEN mutations may impede immune inﬁltration .
To investigate the resistance mechanism of PTEN mutations further, they inspected the single-cell RNA-sequencing proﬁles across 9000 cells in three GBM patients, including one PTEN-mutant sample. They found that cells associated with immune signatures were signiﬁcantly enriched in the PTEN-mutant tumor. They further identiﬁed three major types of cell subpopulations: tumor cells with migration markers (CD44+), actively proliferating tumor cells, and microglia. As expected, the immunosuppressive signature was signiﬁcantly associated with the CD44+ tumor cell subpopulations in the PTENmutant tumor, indicating that PTEN mutations may play a crucial role in the immune In summary, this study suggests that microenvironment and inﬂuence resis- PTEN mutations are signiﬁcantly enriched tance to immunotherapy. in nonresponders to anti-PD-1 inhibitors, offering a potential personalized immunoVia analyzing bulk RNA sequencing pro- therapy approach for GBM. By integrating ﬁles of GBM samples from TCGA, Zhao bulk RNA-seq and published single cell and colleagues showed that PTEN muta- RNA-seq data analysis, the authors furtions are signiﬁcantly correlated with the ther showed that PTEN mutations may forkhead box P3 (FOXP3, a key protein lead to a distinct immunosuppressive
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Phosphatase domain MoƟf 4
TI loop K147 R173 Y68
CBR3 loop L247
Figure 1. A Proposed Model Illustrating the Somatic PTEN Mutation-Associated Resistance to Immunotherapy in Patients with Glioblastoma (GBM). Individual red spheres correspond to PTEN mutations at given residue positions. Mutations occur in both the phosphatase domain (white) and C2 domain (grey) of wild-type PTEN (PDB ID: 1D5R). Critical loops and motifs are labelled as follows: ATP-B binding motif (residues 60–73, pink); WPD loop (residues 88–98, brown); P loop (residues 123–131, cyan); TI loop (residues 160–171, purple); Motif 1 (residues 169–180, blue); Motif 2 (residues 250–259, red); Motif 3 (residues 264–276, orange); Motif 4 (residues 321–334, yellow); CBR3 loop (residues 260–269, ice blue); and domain linker (residues 185–191, green). The protein structural image was generated utilizing Visual Molecular Dynamics Software version 1.9.3. Abbreviations: PD-1, Programed cell death 1; PD-L1: protein programmed death-ligand 1.
microenvironment, which helps to explain the resistance mechanisms to immunotherapy. This observation is consistent with two recent studies showing that loss of PTEN was associated with resistance to immunotherapy in metastatic uterine leiomyosarcoma  and melanoma . This somatic data is also consistent with observations of T and B cell dysfunction in subjects with heterozygous germline PTEN mutations .
Several speciﬁc questions were not explored in this study . Although multiple PTEN mutations were signiﬁcantly enriched in nonresponders, interrogation of the underlying molecular consequences of immunosuppressive microenvironments by PTEN mutations in GBM remains unclear. These observations are aligned with PTEN antiangiogenesis signaling and inhibition of the NF-kB-JAK/ STAT (nuclear factor kappa-light-chain-
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enhancer of activated B cells-Janus kinase/signal transducer and activator of transcription) pathways. Indeed, increased vascular endothelial growth factor A (VEGFA) and STAT3, accompanied by T cell inﬁltration in the tumor-associated stroma, have been found in patients resistant to anti-PD-1/PD-L1 therapy in PTEN-null melanomas and sarcomas [5,6]. PTEN-Long (a speciﬁc translational variant of PTEN) translates
upstream from canonical PTEN; knockdown of PTEN-Long results in decreased TNF-a (tumor necrosis factor alpha) and IL-6 in the microenvironment . PD1/PD-L1 blockade prompts PD-1+ macrophages to produce IL-6 in the tumor microenvironment, at least in a murine melanoma model . Thus, lack of PTEN-Long would prevent the IL-6 response and enhance resistance to immunotherapy. Thus, we can postulate that PD-1/PD-L1 blockade together with PTEN-Long might be a suitable combination therapy to diminish resistance to immunotherapy.
Phase III trial showed that combined ave- Disclaimer Statement lumab (an FDA-approved PD-L1 mono- The authors have declared that no conﬂict of interest clonal antibody) and axitinib [an FDA- exists. approved multiple-tyrosine kinase inhibi- 1Genomic Medicine Institute, Lerner Research Institute, tor (including VEGF receptors 1–3)] leads Cleveland Clinic, Cleveland, OH 44195, USA 2 to signiﬁcantly longer progression-free Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve survival compared with sunitinib (an University, Cleveland, OH 44195, USA FDA-approved multitargeted receptor 3Case Comprehensive Cancer Center, Case Western tyrosine kinase inhibitor) alone in patients Reserve University School of Medicine, Cleveland, OH 44106, USA with advanced renal-cell carcinoma 4Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH (NCT02684006). Thus, PD-1 inhibitors 44195, USA 5 combined with antiangiogenic therapy Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, may overcome the resistance triggered Cleveland, OH 44106, USA by PTEN mutations in GBM; this warrants further testing in preclinical studies or *Correspondence: [email protected]
(F. Cheng) and [email protected]
(C. Eng). clinical trials. This approach seems plauhttps://doi.org/10.1016/j.molmed.2019.03.003 sible, as PTEN loss or dysfunction upregulates angiogenesis. In addition, Zhao © 2019 Elsevier Ltd. All rights reserved. and colleagues found that mutations in the mitogen-activated protein kinase References (MAPK) pathways [such as BRAF (v-Raf 1. Cheng, F. et al. (2019) Personal mutanomes meet modern oncology drug discovery and precision health. Pharmacol. murine sarcoma viral oncogene homolog Rev. 71, 1–19 B) and PTPN11 (tyrosine-protein phos2. Zhao, J. et al. (2019) Immune and genomic correlates of phatase non-receptor type 11)] are response to anti-PD-1 immunotherapy in glioblastoma. Nat. Med. 25, 462–469 signiﬁcantly enriched in GBM responders. 3. Yehia, L. et al. (2019) PTEN-opathies: from biological Taken together, a combination of PD-1 insights to evidence-based precision medicine. J. Clin. Invest. 129, 452–464 inhibitors with approved MAPK pathway (also downstream of PTEN) inhibitors may 4. Chen, H.H. et al. (2017) Immune dysregulation in patients with PTEN hamartoma tumor syndrome: analysis of offer potential strategies for treating FOXP3 regulatory T cells. J. Allergy Clin. Immunol. 139, 607–620 PTEN-mutant patients with GBM. In 5. George, S. et al. (2017) Loss of PTEN is associated with summary, this study offers potential resistance to anti-PD-1 checkpoint blockade therapy in metastatic uterine leiomyosarcoma. Immunity 46, 197– approaches for development of person204 alized immunotherapy in GBM, although 6. Peng, W. et al. (2016) Loss of PTEN promotes resistance further clinical trials and replicated studies to T cell-mediated immunotherapy. Cancer Discov. 6, 202–216 are warranted.
Two independent studies published in the same issue reported that GBM patients could potentially beneﬁt from anti-PD-1 immunotherapies [9,10]. Thus, in the future, replicated studies that examine whether PTEN mutations are associated with resistance to immunotherapy are warranted in independent GBM cohorts and other cancer types. Finally, overall PD-1 inhibitors do not offer a survival beneﬁt for GBM patients and RNA expression of the protein programmed death-ligand 1 (PD-L1 encoded by CD274) is not signiﬁcantly different between responsive and nonresponsive tumors. Development of clinically actionable biomarkers and novel immunotherapeutic strategies (such as combination regimens) using novel technologies are a pressing need. Previous studies have suggested that immunosuppressive Acknowledgments cytokines (such as VEGF) are signiﬁcantly We thank Iris Smith for preparing the protein structural elevated in tumors with PTEN loss. visualization of PTEN somatic mutations. This work Several clinical trials are investigating was supported by the National Heart, Lung, and the clinical beneﬁts of combined antian- Blood Institute of the National Institutes of Health under Award Number K99HL138272 and giogenic agents (anti-VEGF therapy) R00HL138272 to F.C. C.E. is the Sondra J. and and PD-1 inhibitors in several cancer Stephen R. Hardis Endowed Chair in Cancer Genotypes, such as metastatic kidney cancer mic Medicine at the Cleveland Clinic and an ACS (NCT02210117) and non-small cell lung Clinical Research Professor. cancer (NCT02443324). For example, a
7. Wang, H. et al. (2019) Expression of PTEN-long nephritis and its effect on renal inﬂammation. Exp. Ther. Med. 17, 1405–1411 8. Tsukamoto, H. et al. (2018) Combined blockade of IL6 and PD-1/PD-L1 signaling abrogates mutual regulation of their immunosuppressive effects in the tumor microenvironment. Cancer Res. 78, 5011–5022 9. Schalper, K.A. et al. (2019) Neoadjuvant nivolumab modiﬁes the tumor immune microenvironment in resectable glioblastoma. Nat. Med. 25, 470–476 10. Cloughesy, T.F. et al. (2019) Neoadjuvant anti-PD-1 immunotherapy promotes a survival beneﬁt with intratumoral and systemic immune responses in recurrent glioblastoma. Nat. Med. 25, 477–486
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