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Evading the STING: LKB1 Loss Leads to STING Silencing and Immune Escape in KRAS-Mutant Lung Cancers Carminia Maria Della Corte and Lauren Averett Byers

Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. Corresponding Author: Lauren Averett Byers, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 0432, Houston, TX 77030. Phone: 713-792-6363; Fax: 713-792-1220; E-mail: lbyers@mdanderson.org doi: 10.1158/2159-8290.CD-18-1286 ©2019 American Association for Cancer Research.

Summary: Mutations in STK11 (LKB1) are a major cause of primary resistance to immunotherapy in non–small cell lung cancer. Kitajima and colleagues dissect the underlying mechanism of this immune-resistant pheno-type, demonstrating that LKB1 loss leads directly to suppression of stimulator of interferon genes (STING) and insensitivity to cytoplasmic double-strand DNA detection. Therapies that reactivate LKB1 or theSTING pathway may boost anticancer immune response in cancers with resistance to immune-checkpoint blockade.

See related article by Kitajima et al., p. 34 (6).

Lung adenocarcinoma accounts for the majority of non–small cell lung cancers (NSCLC) and includes a variety of molecular subtypes with distinct therapeutic vulnerabilities and resistance patterns. The most frequent oncogenic driver in lung adenocarcinoma is KRAS mutation, which is found in 25% to 30% of cases. However, previous attempts to develop KRAS-targeted therapy have failed, in large part because of their heterogeneity—a consequence of concomitant co-mutations. In a previous landmark study led by Skoulidis and colleagues, our group described molecular features of two key subgroups of KRAS -driven lung adenocarcinoma, those with comutations in TP53 (KP) or in STK11 /LKB1 (KL; ref. 1 ). The KP and KL subtypes were especially different in terms of metabolic and immune profi les, with KL tumors characterized by downregu-lation of PD-L1 and other immune-checkpoint genes ( Fig. 1A ).

LKB1 acts as a critical regulator of cellular metabolism and energy sensing by activating AMP kinase (AMPK). Once acti-vated, AMPK inhibits mTOR signaling. Loss of LKB1 increases serine utilization and synthesis of S-adenosyl methionine (SAM), a substrate of DNMT1, EZH2, and other epigenetic silencing enzymes ( 2 ). Thus, STK11 mutation (and the con-sequent LKB1 loss) directly induces epigenetic changes and confers a proliferative advantage to cancer cells.

Among lung adenocarcinomas, STK11- mutant tumors are “immune-cold” ( Fig. 1A ), having low or absent PD-L1 levels, ineffective T-cell infi ltration, and preferential production of myeloid-recruiting chemokines, such as IL6 ( 1, 3 ). Earlier this year, Skoulidis and colleagues further demonstrated that STK11 /LKB1 inactivation is a major genetic driver of primary resistance to anti–PD-1 agents ( 4 ), and that patients harboring

STK11 /LKB1 alterations have shorter progression-free and overall survival after anti–PD-1 treatment ( Fig. 1B ). This fi nding was further validated by Kunkel and colleagues, who showed that STK11 /LKB1 mutation in nonsquamous NSCLC predicted worse outcomes after treatment with anti–PD-L1 (durvalumab) plus/minus anti-CTLA4 (tremelimumab) from two clinical studies of previously treated patients with NSCLC ( 5 ). Together, these data strongly support STK11 /LKB1 alter-ations as a marker of immune-checkpoint blockade resist-ance. However, a better understanding of the mechanisms regulating immune suppression in KL lung adenocarcinomas is critical for developing effective combination strategies that reestablish antitumor immunity.

In this issue, Kitajima and colleagues explore the mecha-nism by which STK11 /LKB1 inactivation leads to immune escape ( 6 ). They fi nd that LKB1 loss results in insensitiv-ity to cytoplasmic double-strand DNA (dsDNA) detection through transcriptional silencing of stimulator of interferon genes (STING; encoded by TMEM173 ). The STING pathway is a critical component of the innate immune system and is responsible for sensing cytosolic DNA from pathogens (e.g., viruses or bacteria) or our own cells (e.g., cancer). In cancer cells, STING is activated by dsDNA released from the nucleus and mitochondrial compartments into the cytosol, and represents a key, intrinsic tumor-sensing mechanism.

Using gene-expression data to compare KL and KP lung adenocarcinomas, the authors found that KL lung adeno-carcinomas from The Cancer Genome Atlas (TCGA) cohort and cell line models exhibit downregulation of the type I IFN signaling signature, including the TMEM173 (STING) gene (a common upstream regulator of type I IFN). They also observed upregulation of oxidative stress signaling, as expected for the mitochondrial impairment due to LKB1 dysfunction. They then validated these fi ndings by IHC and western blot, showing that LKB1 loss at the protein level is associated with low or absent STING protein in KL tumors and cell lines (but intact cGAS levels). In contrast, KP mod-els consistently expressed relatively higher levels of STING protein at baseline. However, expression of STING could be directly inhibited in KP models by LKB1 deletion using

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CRISPR/Cas9 silencing. Furthermore, STING expression in KL cells could be rescued by LKB1 reconstitution in cells with low levels of STING, but not in those without any detectable STING. Together, these results support the direct transcrip-tional regulation of STING by LKB1.

Next, the authors elegantly connected the role of LKB1 as an epigenetic regulator to their observations. By exploring the functional role of the TMEM173 (STING) promoter, the authors found that KL cells exhibit high activity of DNA methyltransferase DNMT1 and EZH2, which can silence STING transcription by promoter methylation. Interestingly, DNMT1 and EZH2 are both substrates of SAM, which is increased by LKB1; and treatment with specific inhibitors of DNMT1 (e.g., decitabine) and/or EZH2 restored both SAM levels and STING expression.

The authors then tested the effect of LKB1 reconstitu-tion on DNA sensing by stimulating cells with poly (dA:dT) (which mimics cytosolic dsDNA). They found that LKB1 reconstitution in STINGlo KL cells restored STING down-stream signaling. The activation of the STING intracellular phosphorylation cascade through TBK1 and IRF3 led to

the release of the immune inflammatory cytokines IFNβ, CXCL10, CCL5, GM-CSF, CCL3, and IL1α and suppression of IL6, as well as the activation of STAT1 (which stimulates PD-L1 expression; Fig. 1C). Of note, in STINGabsent KL cells, the addition of DNMT inhibitor is necessary to restore all signals, further supporting that LKB1 loss is a direct cause of suppressed antitumor immunity and low PD-L1 expression. In addition, the authors confirm that transient activation of STING prompted via poly (dA:dT) and pretreatment with a DNMT inhibitor prevents tumor formation in STINGabsent KL xenografts, although they were not able to extend these data in terms of sustained STING activation, because avail-able STING agonists do not penetrate into tumor cells.

Finally, given the mitochondrial dysfunction of KL cancer cells, Kitajima and colleagues investigated the contribution of nuclear and mitochondrial DNA (mtDNA) to the pool of cytosolic dsDNA. They found that cytosolic leakage of mtDNA, more than nuclear dsDNA, is essential to induce STAT1 activation. Furthermore, leakage of mtDNA is a key mediator of aberrant STING signaling in KL cells and results in a selection pressure for these cells to silence STING.

Figure 1.  A, Lung adenocarcinomas with both KRAS and STK11 mutations (KL) have lower expression immune marker genes than those with KRAS and TP53 mutations (KP). PD-L1 protein expression, detected by IHC, is also lower in KL tumors. Adapted from Skoulidis et al. (4). B, LKB1-deficient (STK11/LKB1-mutant and/or LKB1 H-score = 0; N = 61) patients with KRAS-mutant lung adenocarcinoma show significantly shorter median overall survival (mOS) and median progression-free survival (mPFS) defined in months (m) after PD-1 blockade. Adapted from Skoulidis et al. (4). C, Working model for the mechanism of STING silencing by LKB1, as demonstrated by Kitajima et al. (6). Reconstitution of LKB1 signaling or direct activation of STING by STING enhancers, along with induction of DNA damage, leads to increased antitumor innate immunity signals and higher PD-L1 expression. In particular, in LKB1-proficient cancer cells, the reactivation of AMPK inhibits methylation of the STING promoter by methyltransferases DNMT1 and EZH2, thus reactivating STING transcription; contemporarily, the release of cytosolic DNA, more from mitochondrial compartment, contributes to STING pathway activation by the DNA sensor cGAS. D, Top, percentage of TCGA patients with lung adenocarcinoma harboring mutations in KRAS and STK11 (KL), KRAS and TP53 (KP), KRAS-only, STK11, TP53, and others. STK11 mutation is observed in 14% of lung adenocarcinomas, of which 7% co-occur with a KRAS mutation. Bottom, frequency of STK11 and TP53 mutations in patients with KRAS-mutant and wild-type (WT) lung adenocarcinoma: Significantly more STK11 mutations are seen in the KRAS-mutant population (37%) than in the KRAS WT population (10%; P < 0.001).

LKB1 LKB1

P-AMPK P-AMPK

mTOR mTOR

DNMT1 EZH2 DNMT1EZH2

SAM SAM

+

Expression

HAVCR2

PDCD1LG2CD274 (PD-L1)

LAG3PDCD1

ICOSCTLA4

STK11 mutation correlates with low expression of immune genes and PD-L1A

C D

KL KP

–2 –1 0 1 2

CD274 (PD-L1) IHC

LKB1-mutant cell LKB1-proficient cells

Mitochondria Mitochondria

mtDNA (mitochondrial dsDNA) nDNA (nuclear dsDNA)

LKB1 reconstitutionor

STING enhancersInflammatorycytokines

STINGSTING

PD–L1

cGASNucleus Nucleus

KP1 KL1

KP2 KL2

p5341%

Other22%

KP11%

Lung adenocarcinoma

KL7%

KRAS-only12%

KRAS

mutant

(30%)

STK117%

KRAS mut KRAS WT0%

10%20%30%40%50%60%70%80%90%

100%

100

90

80

;STK11/LKB1 def

;STK11/LKB1 prof

;STK11/LKB1 def

;STK11/LKB1 prof KRAS

KRAS

KRAS

KRAS

70

60

50

40

30

20

10

0

100

90

80

70

60

50

40

30

20

10

00 6

HR for death 2.03(95% CI, 1.13–3.65)P = 0.016, log-rank test

HR for disease progressionor death 1.80(95a% Cl, 1.15–2.82)P = 0.0094, log-rank test

12 18 24 30Months Months

36 42 48 0 3 6 9 12 15 18 21 24

Ove

rall

surv

ival

(%

)

Pro

gres

sion

–fre

e su

rviv

al (

%)

38(0)61(0)

28(3)29(5)

17(8)12(14)

2(20)5(19)

1(21)3(20)

1(21)2(21)

1(21)2(21)

0(22)1(22)

0(22)0(23)

38(0)61(0)

18(2)14(3)

9(3)7(4)

5(5)5(4)

3(7)3(5)

1(8)2(5)

1(8)1(5)

0(8)0(5)

0(8)0(5)

Group mOS

8.0 m16.0 m

Group

;STK11/LKB1 def;STK11/LKB1 def

;STK11/LKB1 prof;STK11/LKB1 prof

KRASKRAS

KRASKRAS

mPFS

1.8 m3.3 m

Other Other

TP53

STK11

STK11

TP53

LKB1 loss is predictive of resistance to PD-1 blockadeB

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Taken together, this work provides strong evidence to sup-port STAT1 as the main mediator of STING-dependent cyto-toxicity and PD-L1 expression in lung adenocarcinomas with KRAS/STK11 (LKB1) co-mutation. This discovery has key therapeutic implications for patients with KL lung adenocar-cinoma, an aggressive lung cancer subtype that is refractory to existing standard-of-care immune-checkpoint inhibitors. Indeed, in KL tumors, where STING is inactivated by LKB1, all downstream signals of antitumor immunity, including IRF3 and STAT1, are silenced and other protumorigenic sig-nals (mainly mediated by NF-KB) sustain tumor progression. In summary, Kitajima and colleagues demonstrate for the first time that LKB1 is a key regulator of STING transcription, thus revealing a novel mechanism of immune resistance of KL lung adenocarcinoma.

The ability to convert a KL immune-resistant, noninflamed tumor into an immune-sensitive, inflamed tumor with LKB1 reconstitution or the use of STING agonists is supported by the in vitro model data. However, this is likely to prove more difficult in patients. Readily available therapeutic options that may increase the release of cytosolic DNA and STING activation include DNA-damaging therapies such as radio-therapy, chemotherapy, or DNA damage response (DDR) inhibitors (e.g., PARP inhibitors; ref. 7). However, direct stim-ulation of innate immunity through STING agonists is being actively pursed both in vivo and in clinical trials. For example, synthetic cyclic dinucleotides that mimic the STING ligand cGAMP are being explored as an approach to activate STING. One of these is MK-1454, which binds directly to STING in the cytosol. Preliminary data from the phase I trial of MK-1454 with pembrolizumab (NCT03010176) showed a safe tolerability profile and encouraging efficacy, with serum levels of STING-dependent cytokines increased posttreat-ment, confirming STING activation (8). However, MK-1454 requires intratumor injection, which poses obvious chal-lenges in clinical practice (especially in patients without easily accessible tumors or with multifocal disease).

In November 2018, development of the first intravenous STING agonist (made up of two linked amidobenzimidazole compounds) with strong antitumor activity in a colon cancer model was reported by Ramanjulu and colleagues (9). Another strategy to target the STING pathway is by targeting the metabolism of nucleotides and stabilization of cGAMP (nor-mally produced by cGAS in response to cytosolic DNA and then detected directly by STING). The oral agent MV-626 is a potent inhibitor of ectonucleotide pyrophosphatase/phos-phodiesterase 1 (ENPP1; the phosphodiesterase of cGAMP) and has preclinical activity in combination with radiotherapy and anti–PD-L1 (10). Based on promising preclinical data, together with rapidly advancing options for STING targeting in the clinic, combinations of STING enhancers (e.g., STING agonists or ENPP1 inhibitors) with immunotherapy warrant further testing in KL lung adenocarcinomas. In addition, find-ings from Kitajima and colleagues support further investiga-tion into the role of inhibitors of DNA methyltransferase or DDR, combined with STING enhancers or immunotherapy.

Finally, although this study focused on KRAS/STK11 comu-tated lung adenocarcinoma, it is likely that the findings have broader applications for overcoming immunotherapy resist-ance in other cancers with STK11/LKB1 mutation (Fig. 1D).

For example, LKB1 loss is also found in lung cancers without KRAS mutations. Beyond lung cancer, STK11 sporadic muta-tions are found in cervical, breast, intestinal, testicular, pan-creatic, and skin cancers; and germline STK11 mutations are associated with Peutz–Jeghers syndrome, a genetic disorder characterized by risk of various neoplasms, including intes-tinal and skin tumors. In conclusion, therapeutic strategies that can reactivate the LKB1 or STING pathway hold prom-ise as a new way to boost anticancer immune response and to sensitize tumors that are resistant to anti–PD-1/PD-L1 agents in NSCLC and other cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThis work was supported by NIH/NCIR01-CA207295 (L.A. Byers),

NIH/NCIU01-CA213273 (L.A. Byers), NIH/NCIP5-CA070907 (L.A. Byers), and NIH/NCIP30-CA01667 (L.A. Byers), and through the gen-erous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shots Program (L.A. Byers).

Published online January 9, 2019.

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8. Harrington KJ, Brody J, Ingham M, Strauss J, Cemerski S, Wang M, et  al. Preliminary results of the first-in-human (FIH) study of MK-1454, an agonist of stimulator of interferon genes (STING), as monotherapy or in combination with pembrolizumab (pembro) in patients with advanced solid tumors or lymphomas. Ann Oncol 2018;29(suppl_8):LBA15.

9. Ramanjulu JM, Pesiridis GS, Yang J, Concha N, Singhaus R, Zhang SY, et  al. Design of amidobenzimidazole STING receptor agonists with systemic activity. Nature 2018; Nov 7 [Epub ahead of print].

10. Baird J, Dietsch G, Florio V, Gallatin M, Knox C, Odingo J, et  al. MV-626, a potent and selective inhibitor of ENPP1 enhances STING activation and augments T-cell mediated anti-tumor activity in vivo. J Immunother Cancer 2018;6(Suppl 1):115, P410.

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2019;9:16-18. Cancer Discov   Carminia Maria Della Corte and Lauren Averett Byers  Immune Escape in KRAS-Mutant Lung CancersEvading the STING: LKB1 Loss Leads to STING Silencing and

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