Oncogenic RAS Signaling Promotes Tumor Immunoresistance by Stabilizing PD-L1 mRNA

•Oncogenic RAS signaling via MEK increases PD-L1 expression•RAS regulates PD-L1 through AU-rich elements (AREs) in the 3′ UTR of PD-L1 mRNA•The ARE-binding protein tristetraprolin (TTP) negatively regulates PD-L1 expression•Restoration of tumor cell TTP activity enhances anti-tumor immunity The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3′ UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers. The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3′ UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers. Therapeutic antibodies blocking the coinhibitory PD-1 pathway by targeting PD-L1 (programmed death 1 ligand 1, also known as B7-H1 or CD274) or its receptor, PD-1, have caused striking regressions in several malignancies in which RAS mutations are frequent driver events, including non-small cell lung cancer (NSCLC) (Herbst et al., 2014Herbst R.S. Soria J.C. Kowanetz M. Fine G.D. Hamid O. Gordon M.S. Sosman J.A. McDermott D.F. Powderly J.D. Gettinger S.N. et al.Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients.Nature. 2014; 515: 563-567Crossref PubMed Scopus (3695) Google Scholar, Topalian et al., 2012Topalian S.L. Hodi F.S. Brahmer J.R. Gettinger S.N. Smith D.C. McDermott D.F. Powderly J.D. Carvajal R.D. Sosman J.A. Atkins M.B. et al.Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.N. Engl. J. Med. 2012; 366: 2443-2454Crossref PubMed Scopus (9275) Google Scholar) and mismatch-repair-deficient colorectal cancer (Le et al., 2015Le D.T. Uram J.N. Wang H. Bartlett B.R. Kemberling H. Eyring A.D. Skora A.D. Luber B.S. Azad N.S. Laheru D. et al.PD-1 blockade in tumors with mismatch-repair deficiency.N. Engl. J. Med. 2015; 372: 2509-2520Crossref PubMed Scopus (6109) Google Scholar). PD-L1 is critical for limiting autoimmune-related damage to normal tissues in the context of chronic inflammation but is also aberrantly upregulated on cancer cells in order to evade immune destruction (Pardoll, 2012Pardoll D.M. The blockade of immune checkpoints in cancer immunotherapy.Nat. Rev. Cancer. 2012; 12: 252-264Crossref PubMed Scopus (8861) Google Scholar). As anti-PD-1 pathway immunotherapies are effective in only a minority of cancer patients (Topalian et al., 2012Topalian S.L. Hodi F.S. Brahmer J.R. Gettinger S.N. Smith D.C. McDermott D.F. Powderly J.D. Carvajal R.D. Sosman J.A. Atkins M.B. et al.Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.N. Engl. J. Med. 2012; 366: 2443-2454Crossref PubMed Scopus (9275) Google Scholar), there is a great need for reliable biomarkers of patient response. To what degree tumor PD-L1 expression is prognostic of patient response to PD-1 pathway blockade remains contentious. Recent clinical trials of the anti-PD-1 antibody nivolumab report that tumor cell PD-L1 expression correlates with response to nivolumab in non-squamous but not the squamous subtype of NSCLC (Borghaei et al., 2015Borghaei H. Paz-Ares L. Horn L. Spigel D.R. Steins M. Ready N.E. Chow L.Q. Vokes E.E. Felip E. Holgado E. et al.Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer.N. Engl. J. Med. 2015; 373: 1627-1639Crossref PubMed Scopus (6773) Google Scholar, Brahmer et al., 2015Brahmer J. Reckamp K.L. Baas P. Crinò L. Eberhardt W.E. Poddubskaya E. Antonia S. Pluzanski A. Vokes E.E. Holgado E. et al.Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer.N. Engl. J. Med. 2015; 373: 123-135Crossref PubMed Scopus (6236) Google Scholar). Notably, non-squamous NSCLC patients with KRAS mutations benefited from nivolumab therapy in terms of overall survival, whereas KRAS wild-type patients did not (Borghaei et al., 2015Borghaei H. Paz-Ares L. Horn L. Spigel D.R. Steins M. Ready N.E. Chow L.Q. Vokes E.E. Felip E. Holgado E. et al.Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer.N. Engl. J. Med. 2015; 373: 1627-1639Crossref PubMed Scopus (6773) Google Scholar). Response rate and progression-free survival was increased in NSCLC patients treated with pembrolizumab in cases where at least 50% of tumor cells were positive for PD-L1 (Garon et al., 2015Garon E.B. Rizvi N.A. Hui R. Leighl N. Balmanoukian A.S. Eder J.P. Patnaik A. Aggarwal C. Gubens M. Horn L. et al.KEYNOTE-001 InvestigatorsPembrolizumab for the treatment of non-small-cell lung cancer.N. Engl. J. Med. 2015; 372: 2018-2028Crossref PubMed Scopus (4358) Google Scholar). In this patient cohort, KRAS mutant tumors were more frequently PD-L1 positive than KRAS wild-type tumors. The success of immune-checkpoint blockade is dependent on the immunogenicity of the tumor (Gubin et al., 2014Gubin M.M. Zhang X. Schuster H. Caron E. Ward J.P. 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Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.Science. 2015; 348: 124-128Crossref PubMed Scopus (5531) Google Scholar), so one possible confounding factor in the use of tumor PD-L1 as a biomarker for response is the uncoupling of tumor PD-L1 expression from tumor immunogenicity. It is therefore critical to understand the signaling pathways that dictate tumor cell PD-L1 expression. The inflammatory cytokine IFN-γ is the best-characterized stimulus for PD-L1 expression, but several studies suggest that cell-intrinsic oncogenic signaling can also promote PD-L1 expression in cancer cells through epidermal growth factor receptor (EGFR), the transcription factor MYC, and the kinase AKT (Akbay et al., 2013Akbay E.A. Koyama S. Carretero J. Altabef A. Tchaicha J.H. Christensen C.L. Mikse O.R. Cherniack A.D. Beauchamp E.M. 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The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition.Clin. Cancer Res. 2013; 19: 598-609Crossref PubMed Scopus (356) Google Scholar) and acute myeloid leukemia (Berthon et al., 2010Berthon C. Driss V. Liu J. Kuranda K. Leleu X. Jouy N. Hetuin D. Quesnel B. In acute myeloid leukemia, B7-H1 (PD-L1) protection of blasts from cytotoxic T cells is induced by TLR ligands and interferon-gamma and can be reversed using MEK inhibitors.Cancer Immunol. Immunother. 2010; 59: 1839-1849Crossref PubMed Scopus (125) Google Scholar) have indicated that MEK signaling is involved in upregulation of PD-L1 in some tumor cell lines, but the molecular basis of this regulation remains poorly defined. Separately, genetic rearrangements in the 3′ UTR of CD274 (encoding PD-L1) have been found in a multitude of different cancers at low frequency and are associated with massively increased expression of tumor PD-L1 (Kataoka et al., 2016Kataoka K. Shiraishi Y. Takeda Y. Sakata S. Matsumoto M. Nagano S. Maeda T. Nagata Y. Kitanaka A. Mizuno S. et al.Aberrant PD-L1 expression through 3′-UTR disruption in multiple cancers.Nature. 2016; 534: 402-406Crossref PubMed Scopus (422) Google Scholar). These results imply that control of PD-L1 expression through the CD274 3′ UTR might contribute to immune escape in human cancers, although the underlying mechanisms of post-transcriptional regulation responsible for this effect are unclear. In this report, we reveal that tumor cell PD-L1 expression can be driven by oncogenic RAS pathway activation by a mechanism involving post-transcriptional regulation of the stability of PD-L1 mRNA. This provides a direct mechanism whereby RAS signaling in tumor cells can provide protection from attack by the immune system. We tested the potential role of oncogenic RAS signaling in the regulation of PD-L1 expression in human epithelial cells using ER-RASG12V fusion constructs, which allow for the induction of oncogenic RAS activity with 4-hydroxytamoxifen (4-OHT) (Molina-Arcas et al., 2013Molina-Arcas M. Hancock D.C. Sheridan C. Kumar M.S. Downward J. Coordinate direct input of both KRAS and IGF1 receptor to activation of PI3 kinase in KRAS-mutant lung cancer.Cancer Discov. 2013; 3: 548-563Crossref PubMed Scopus (129) Google Scholar). As expected, addition of 4-OHT led to the rapid activation of oncogenic KRAS signaling through MEK and PI3K (Figure 1A) and coincided with induction of MYC mRNA and CCND1 mRNA (encoding cyclin D1) in an immortalized human pneumocyte cell line derived from type II cells (Figure 1B; Kemp et al., 2008Kemp S.J. Thorley A.J. Gorelik J. Seckl M.J. O’Hare M.J. Arcaro A. Korchev Y. Goldstraw P. Tetley T.D. Immortalization of human alveolar epithelial cells to investigate nanoparticle uptake.Am. J. Respir. Cell Mol. Biol. 2008; 39: 591-597Crossref PubMed Scopus (117) Google Scholar). PD-L1 mRNA was rapidly increased following stimulation of oncogenic KRAS signaling with 4-OHT, resulting in a 6-fold induction of mRNA expression after 3 hr (Figure 1B). By way of comparison with known regulators, stimulation with IFN-γ led to increases in PD-L1 mRNA in excess of 10-fold after 3 hr and both KRAS activation and IFN-γ stimulation dramatically increased PD-L1 protein expression at the cell surface after 48 hr (Figure 1C). Oncogenic HRAS signaling was also capable of inducing PD-L1 mRNA and protein expression in the immortalized breast epithelial cell line MCF10A and the KRAS wild-type colon carcinoma cell line HKE-3 (Figures S1A and S1B), implying that induction of PD-L1 expression by RAS is not a tissue-specific or RAS-isoform-specific phenomenon. The induction of PD-L1 protein was most striking in ER-HRASG12V MCF10A cells, perhaps reflecting the low basal expression of PD-L1. Chronic RAS activation for 4 days led to more profound increases in PD-L1 protein, whereas shorter-term activation resulted in modest inductions of PD-L1 expression (Figure S1B). Importantly, 4-OHT did not induce PD-L1 expression in parental cell lines lacking ER-RAS constructs (Figure S1C). Direct inhibition of KRAS signaling with the KRASG12C-specific inhibitor ARS853 (Lito et al., 2016Lito P. Solomon M. Li L.S. Hansen R. Rosen N. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.Science. 2016; 351: 604-608Crossref PubMed Scopus (397) Google Scholar, Patricelli et al., 2016Patricelli M.P. Janes M.R. Li L.S. Hansen R. Peters U. Kessler L.V. Chen Y. Kucharski J.M. Feng J. Ely T. et al.Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state.Cancer Discov. 2016; 6: 316-329Crossref PubMed Scopus (446) Google Scholar) in lung and colorectal cancer cell lines harboring KRASG12C mutations led to reductions in PD-L1 mRNA expression, but not in the KRASG12S A549 control lung cancer cell line (Figures 1D and 1E). Moreover, ARS853 treatment led to significant reductions in PD-L1 surface protein expression in the KRAS mutant lung cancer cell line H358 (Figure 1F). To dissect which downstream effectors of RAS are responsible for regulating PD-L1 expression, we used the specific inhibitors of MEK and pan type I PI3Ks, GSK1120212 (trametinib) and GDC-0941 (pictilisib), respectively (Figure S1D). Notably, MEK and PI3K inhibitors could block RAS-induced expression of PD-L1 protein in ER-KRASG12V type II pneumocytes, either alone or in combination (Figure 1G). MEK inhibition significantly reversed KRAS-mediated PD-L1 mRNA upregulation (Figure 1H), but PI3K inhibition only reduced PD-L1 protein expression, concordant with evidence for AKT signaling increasing PD-L1 expression predominantly through activating translation of the transcript (Parsa et al., 2007Parsa A.T. Waldron J.S. Panner A. Crane C.A. Parney I.F. Barry J.J. Cachola K.E. Murray J.C. Tihan T. Jensen M.C. et al.Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma.Nat. Med. 2007; 13: 84-88Crossref PubMed Scopus (1051) Google Scholar). MEK inhibition, but not PI3K inhibition, reduced PD-L1 mRNA expression in H358 (Figure 1I), H23, and H1792 lung cancer cell lines (Figure S1E). Downstream of MEK, inhibition of ERK1/2 with SCH772984 potently reduced PD-L1 expression in H358 and H23 cells (Figure S1F). Furthermore, PMA, a potent chemical activator of MEK-ERK signaling via protein kinase C stimulation, markedly and rapidly increased PD-L1 expression, an effect that was largely reversed with the inhibition of MEK (Figures 1J and S1G). More extensive analysis of PD-L1 surface expression on multiple KRAS mutant lung cancer cell lines, both human and murine, revealed generally consistent PD-L1 downregulation after MEK and PI3K inhibition, suggesting that this regulatory pathway is of broad significance (Figure S1H). Taken together, these results suggest that oncogenic RAS signaling through MEK and PI3K is sufficient to drive PD-L1 expression. Since RAS signaling has been implicated in reducing the expression of genes involved in the presentation of antigens by MHC class I molecules (Ebert et al., 2016Ebert P.J.R. Cheung J. Yang Y. McNamara E. Hong R. Moskalenko M. Gould S.E. Maecker H. Irving B.A. Kim J.M. et al.MAP kinase inhibition promotes T cell and anti-tumor activity in combination with PD-L1 checkpoint blockade.Immunity. 2016; 44: 609-621Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, El-Jawhari et al., 2014El-Jawhari J.J. El-Sherbiny Y.M. Scott G.B. Morgan R.S. Prestwich R. Bowles P.A. Blair G.E. Tanaka T. Rabbitts T.H. Meade J.L. Cook G.P. Blocking oncogenic RAS enhances tumour cell surface MHC class I expression but does not alter susceptibility to cytotoxic lymphocytes.Mol. Immunol. 2014; 58: 160-168Crossref PubMed Scopus (32) Google Scholar), we analyzed the expression of antigen processing and antigen presentation machinery following oncogenic RAS activation (Figure S1I). As expected, KRAS G12V signaling led to significant decreases in expression of TAP1, TAPBP, as well as HLA-A, HLA-B, HLA-C, and B2M, suggesting that compromised antigen processing and presentation in concert with increases in PD-L1 expression may contribute to an augmented state of immunoresistance in RAS mutant tumor cells. To investigate how RAS-MEK signaling regulates PD-L1 expression, we first asked whether RAS regulates PD-L1 via a transcriptional mechanism. We generated a series of luciferase reporter constructs containing promoter fragments cloned from the human CD274 locus (Figure S2A). In all cases, the physiological stimulus IFN-γ, but not PMA, induced expression of the promoter reporter constructs in H358 cells, a cell line in which endogenous PD-L1 mRNA expression is robustly induced with PMA (Figure 1J). Incorporation of putative enhancer elements (Sumimoto et al., 2016Sumimoto H. Takano A. Teramoto K. Daigo Y. RAS-mitogen-activated protein kinase signal is required for enhanced PD-L1 expression in human lung cancers.PLoS ONE. 2016; 11: e0166626Crossref PubMed Scopus (117) Google Scholar) into the CD274 promoter reporter constructs also failed to confer sensitivity to MAPK activation (Figure S2A), as did including predicted regulatory regions spanning the 5′ of exon 1 (data not shown). Furthermore, none of the reporters showed evidence of decreased expression when H358 cells were treated with MEK inhibitor (data not shown). Therefore, we investigated possible mechanisms of post-transcriptional regulation of PD-L1 expression by RAS. We induced oncogenic KRAS signaling with 4-OHT in ER-KRASG12V type II pneumocytes and concomitantly blocked transcription with actinomycin D. Surprisingly, we found human PD-L1 mRNA to have a short half-life, which was significantly stabilized by the induction of oncogenic KRAS signaling (Figure 2A). Moreover, murine PD-L1 mRNA also had a comparably short half-life, and the stability of the transcript in a Kras mutant, p53-deleted murine lung tumor cell line (KPB6), could be reduced further still when MEK was inhibited (Figure 2B), implicating KRAS-MEK signaling in the stabilization of the labile PD-L1 transcript. Consistently, direct inhibition of oncogenic KRAS signaling with ARS853 also caused reductions in PD-L1 mRNA half-life in H23, H1792, and H358 cells (Figure 2C). However, inhibition of PI3K alone did not result in altered PD-L1 mRNA stability in KPB6 cells (Figure S2B). Common genetic elements conferring mRNA instability include miRNA binding sites and AU-rich elements (AREs) in the 3′ UTR of the transcript. The core motif for AREs is an ATTTA pentamer sequence, but functional AREs are often found in an AU-rich context, conforming to the WWATTTAWW nonamer consensus (where W denotes an A or T) (Zubiaga et al., 1995Zubiaga A.M. Belasco J.G. Greenberg M.E. The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.Mol. Cell. Biol. 1995; 15: 2219-2230Crossref PubMed Scopus (472) Google Scholar) constituting the binding site for several AU-rich element binding proteins (AUBPs), which can subsequently recruit mRNA decay machinery (Lykke-Andersen and Wagner, 2005Lykke-Andersen J. Wagner E. Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1.Genes Dev. 2005; 19: 351-361Crossref PubMed Scopus (371) Google Scholar). For example, a canonical ARE-regulated transcript is TNF, which contains nine pentamer sequences in the human transcript and eight pentamers in the murine transcript. Upon inspection of the 3′ UTR of PD-L1 mRNA, we noted a high number of ARE pentamers. Specifically, out of 14 ATTTA pentamer sequences in the human transcript and 11 in the murine transcript, there were 3 conserved AREs conforming to the nonamer consensus (Figure 2D). We tested the influence of MEK inhibition on the half-life of another unstable transcript, Tusc2 mRNA (tumor suppressor candidate 2, or Fus1), which does not contain AU-rich elements in the 3′ UTR but is targeted by multiple miRNAs (Du et al., 2009Du L. Schageman J.J. Subauste M.C. Saber B. Hammond S.M. Prudkin L. Wistuba I.I. Ji L. Roth J.A. Minna J.D. Pertsemlidis A. miR-93, miR-98, and miR-197 regulate expression of tumor suppressor gene FUS1.Mol. Cancer Res. 2009; 7: 1234-1243Crossref PubMed Scopus (195) Google Scholar). Although Tusc2 mRNA had a similar half-life to PD-L1 mRNA, MEK inhibition did not influence the stability of the Tusc2 transcript (Figure S2C), indicating that the observed post-transcriptional regulation of PD-L1 by MEK may relate to AU-rich elements in the 3′ UTR. Indeed, a transcript containing functional AU-rich elements, Ptgs2 mRNA (Cha et al., 2011Cha H.J. Lee H.H. Chae S.W. Cho W.J. Kim Y.M. Choi H.J. Choi D.H. Jung S.W. Min Y.J. Lee B.J. et al.Tristetraprolin downregulates the expression of both VEGF and COX-2 in human colon cancer.Hepatogastroenterology. 2011; 58: 790-795Crossref PubMed Scopus (1) Google Scholar), displayed a significant reduction in mRNA half-life in response to MEK inhibition (Figure S2C), reminiscent of PD-L1 mRNA. To directly analyze the functional importance of these AREs, we constructed a luciferase reporter containing a fragment of the 3′ UTR of human CD274 containing the last six ATTTA pentamers, including the three conserved nonamer sequences. Mutation of ATTTA pentamers to ATGTA has been shown to increase the expression of ARE-containing mRNAs (Rajagopalan et al., 1995Rajagopalan L.E. Burkholder J.K. Turner J. Culp J. Yang N.S. Malter J.S. Granulocyte-macrophage colony-stimulating factor mRNA stabilization enhances transgenic expression in normal cells and tissues.Blood. 1995; 86: 2551-2558Crossref PubMed Google Scholar, Yang et al., 2004Yang N.S. Wang J.H. Turner J. Molecular strategies for improving cytokine transgene expression in normal and malignant tissues.Gene Ther. 2004; 11: 100-108Crossref PubMed Scopus (4) Google Scholar). Consistent with this, mutating the six ATTTA pentamer sequences to ATGTA increased expression of the PD-L1 3′ UTR luciferase reporter in ER-HRASG12V MCF10A and H358 cells, suggesting that these AREs are functionally relevant for controlling the expression of PD-L1 (Figures 2E and 2F). Stimulation with 4-OHT in ER-HRASG12V MCF10A cells, or PMA in H358 cells, increased expression of the wild-type reporter, whereas the ATGTA mutant reporter was insensitive to these treatments (Figures 2E and 2F). In sum, these data suggest that AREs in the 3′ UTR of PD-L1 mRNA can mediate control of PD-L1 expression by RAS-MEK signaling. To assess which AU-rich element binding proteins (AUBPs) could mediate regulation of PD-L1 expression downstream of RAS signaling, we performed a selected siRNA screen of likely candidate genes, AUF1, KSRP, HuR, and TTP (also known as tristetraprolin or ZFP36), in three RAS mutant lung cancer cell lines (Figures 3A–3C). Knockdown efficiency was verified in each case by qPCR (Figures S3A–S3C). siRNA-mediated knockdown of KSRP and TTP most consistently increased PD-L1 mRNA expression across the cell line panel, with the exception of A427, where knock-down of TTP did not lead to significant increases in PD-L1 mRNA levels. Overexpression of KSRP or TTP was sufficient to significantly decrease PD-L1 expression (Figure 3D) and PD-L1 3′ UTR luciferase reporter expression in H358 cells (Figure 3E), corroborating our results from the siRNA screen and confirming that KSRP and TTP impart their negative regulation of PD-L1 expression through the 3′ UTR. Overexpression of TTP and KSRP together did not result in additive reductions in PD-L1 expression, suggesting that they may regulate PD-L1 through the same mechanism (Figure S3D). Notably, siRNA-mediated knockdown of TTP family members, BRF-1 and BRF-2, was incapable of increasing PD-L1 expression to the extent achieved by silencing TTP expression (Figures S3E and S3F). We confirmed that TTP protein expression was reduced following knock-down in H23 and H358 cells, but this was less clear in A427 cells, which express lower levels of TTP protein (Figure S3G). Deconvolution of siRNA pools targeting TTP showed that multiple siRNAs increased expression of PD-L1 mRNA in H23 and H358 cells (Figure S3H). We further examined the regulation of PD-L1 mRNA by TTP by using TTP wild-type (WT) and TTP knock-out (KO) MEFs. In the TTP KO MEFs, TTP mRNA is expressed but no functional TTP protein can be made due to the introduction of a premature stop codon at the endogenous locus (Lai et al., 2006Lai W.S. Parker J.S. Grissom S.F. Stumpo D.J. Blackshear P.J. Novel mRNA targets for tristetraprolin (TTP) identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts.Mol. Cell. Biol. 2006; 26: 9196-9208Crossref PubMed Scopus (182) Google Scholar, Taylor et al., 1996Taylor G.A. Carballo E. Lee D.M. Lai W.S. Thompson M.J. Patel D.D. Schenkman D.I. Gilkeson G.S. Broxmeyer H.E. Haynes B.F. Blackshear P.J. A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency.Immunity. 1996; 4: 445-454Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). Acute activation of TTP expression with serum temporally coincided with a substantial and transient decrease in PD-L1 mRNA in TTP WT MEFs, but not in the TTP KO MEFs (Figure 3F), with PD-L1 levels recovering to near baseline at 6 hr after serum addition. Moreover, the total absence of functional TTP protein in the TTP KO MEFs increased the half-life of PD-L1 mRNA relative to TTP WT MEFs (Figure 3G). Finally, we generated a KPB6 lung cancer cell line with a tetracycline-inducible TTP transgene (TTP tet-ON). As expected, inducible expression of TTP led to reductions in wild-type PD-L1 3′ UTR luciferase reporter expression, but not of the ATGTA mutant 3′ UTR reporter (Figure 3H). When combined with MEK inhibition, TTP expression more robustly suppressed expression of the wild-type reporter. In sum, these data provide evidence for the negative regulation of PD-L1 mRNA expression by the AUBPs KSRP and TTP. To further investigate whether MEK and TTP regulate PD-L1 via a shared pathway, we silenced TTP expression using siRNAs in the context of MEK inhibition. Knock-down of TTP was largely able to rescue the decrease in PD-L1 expression caused by MEK inhibition (Figure 4A). However, the knockdown of KSRP could not rescue this phenotype, despite profound silencing of expression (Figure S4A). Furthermore, MEK inhibition significantly increased TTP mRNA expression (Figure 4A), and chronic activation of oncogenic KRAS signaling significantly decreased TTP mRNA expression (Figure 4B). Next, we tested whether the RAS pathway regulates the activity of TTP and/or KSRP protein. Crucially, we found that endogenous levels of TTP and KSRP both co-precipitated with PD-L1 mRNA in RNA immunoprecipitation (RNA-IP) reactions from KPB6 mouse lung cancer cells (Figure S4B). TTP also significantly bound to PD-L1 mRNA in H358 cells (Figure 4C). In all cases, the enrichment for the PD-L1 transcript was far greater than that of a control mRNA, GAPDH, which lacks AREs in the 3′ UTR (Figures 4C and S4C). MEK inhibition did not significantly alter the occupancy of TTP or KSRP on PD-L1 mRNA, consistent with RAS regulating the activity of the AUBP, rather than the occupancy on the target mRNA. ERK has been shown to phosphorylate (Taylor et al., 1995Taylor G.A. Thompson M.J. Lai W.S. Blackshear P.J. Phosphorylation of tristetraprolin, a potential zinc finger transcription factor, by mitogen stimulation in intact cells and by mitogen-activated protein kinase in vitro.J. Biol. Chem. 1995; 270: 13341-13347Crossref PubMed Scopus (113) Google Scholar) and negatively regulate TTP activity and expression (Bourcier et al., 2011Bourcier C. Griseri P. Grépin R. Bertolotto C. Mazure N. Pagès G. 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