An Unexpected Partnership: Histone Deacetylase 6 and Glutaminase Inhibition Provide an Opportunity to Overcome Resistance in KRAS and LKB1 Co-mutant Lung Tumors

Immunotherapy has led to improved outcomes for patients with NSCLC.1Gettinger S.N. Horn L. Gandhi L. et al.Overall survival and long-term safety of nivolumab (anti-programmed death 1 antibody, BMS-936558, ONO-4538) in patients with previously treated advanced non-small-cell lung cancer.J Clin Oncol. 2015; 33: 2004-2012Crossref PubMed Scopus (968) Google Scholar,2Gettinger S. Horn L. Jackman D. et al.Five-year follow-up of nivolumab in previously treated advanced non-small-cell lung cancer: results from the CA209-003 study.J Clin Oncol. 2018; 36: 1675-1684Crossref PubMed Scopus (494) Google Scholar However, tumors with inactivation of the serine-threonine kinase LKB1 tumor suppressor (∼20%–30% of NSCLC),3Sanchez-Cespedes M. Parrella P. Esteller M. et al.Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung.Cancer Res. 2002; 62: 3659-3662PubMed Google Scholar, 4Matsumoto S. Iwakawa R. Takahashi K. et al.Prevalence and specificity of LKB1 genetic alterations in lung cancers.Oncogene. 2007; 26: 5911-5918Crossref PubMed Scopus (235) Google Scholar, 5Ding L. Getz G. Wheeler D.A. et al.Somatic mutations affect key pathways in lung adenocarcinoma.Nature. 2008; 455: 1069-1075Crossref PubMed Scopus (2214) Google Scholar, 6Cancer Genome Atlas Research, NetworkComprehensive molecular profiling of lung adenocarcinoma.Nature. 2014; 511: 543-550Crossref PubMed Scopus (3713) Google Scholar are highly resistant to approved immunotherapy approaches.7Skoulidis F. Goldberg M.E. Greenawalt D.M. et al.STK11/LKB1 mutations and PD-1 inhibitor resistance in KRAS-mutant lung adenocarcinoma.Cancer Discov. 2018; 8: 822-835Crossref PubMed Scopus (881) Google Scholar LKB1 is a master kinase upstream of AMPK and together these proteins control cell growth in response to changes in the cellular energy state.8Garcia D. Shaw R.J. AMPK: mechanisms of cellular energy sensing and restoration of metabolic balance.Mol Cell. 2017; 66: 789-800Abstract Full Text Full Text PDF PubMed Scopus (967) Google Scholar, 9Herzig S. Shaw R.J. AMPK: guardian of metabolism and mitochondrial homeostasis.Nat Rev Mol Cell Biol. 2018; 19: 121-135Crossref PubMed Scopus (1700) Google Scholar, 10Trefts E. Shaw R.J. AMPK: restoring metabolic homeostasis over space and time.Mol Cell. 2021; 81: 3677-3690Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Increased proliferation places substantial energy demands on tumors that result in the rewiring of a tumor's metabolic pathways.11Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science. 2009; 324: 1029-1033Crossref PubMed Scopus (10618) Google Scholar, 12Hanahan D. Weinberg R.A. The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22805) Google Scholar, 13Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (45043) Google Scholar Studies in lung cancer have found that NSCLC is a metabolically heterogeneous disease and that inhibiting their rewired metabolic pathways can prove to be a potential therapeutic strategy.14Hensley C.T. Faubert B. Yuan Q. et al.Metabolic heterogeneity in human lung tumors.Cell. 2016; 164: 681-694Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 15Momcilovic M. Bailey S.T. Lee J.T. et al.Targeted inhibition of EGFR and glutaminase induces metabolic crisis in EGFR mutant lung cancer.Cell Rep. 2017; 18: 601-610Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 16Momcilovic M. Bailey S.T. Lee J.T. et al.The GSK3 signaling axis regulates adaptive glutamine metabolism in lung squamous cell carcinoma.Cancer Cell. 2018; 33: 905-921 e5Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar In this issue of the Journal of Thoracic Oncology, Zhang et al.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar evaluate the potential for histone deacetylase 6 (HDAC6) inhibition to serve as a therapeutic approach in metabolically active KRAS-mutant lung adenocarcinomas (LUADs) that have co-occurring LKB1 mutations.6Cancer Genome Atlas Research, NetworkComprehensive molecular profiling of lung adenocarcinoma.Nature. 2014; 511: 543-550Crossref PubMed Scopus (3713) Google Scholar,17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar Of particular clinical importance, the population of NSCLC patients with co-mutations in KRAS and LKB1 have been extremely challenging to manage owing to distinct biology, immune profiles, and therapeutic responses to standard frontline immunotherapy approaches that are well below what has been seen in patients without these alterations.7Skoulidis F. Goldberg M.E. Greenawalt D.M. et al.STK11/LKB1 mutations and PD-1 inhibitor resistance in KRAS-mutant lung adenocarcinoma.Cancer Discov. 2018; 8: 822-835Crossref PubMed Scopus (881) Google Scholar,18Skoulidis F. Byers L.A. Diao L. et al.Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities.Cancer Discov. 2015; 5: 860-877Crossref PubMed Scopus (592) Google Scholar As a result, there is now a renewed interest in murine preclinical studies aimed at identifying novel therapeutic approaches for patients with KRAS and LKB1 co-mutations. Zhang et al.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar studied genetically engineered mouse models with either a KRAS and LKB1 (KL) or KRAS and TP53 (KP) mutant background and found that KL cells had lower oxygen consumption, rates and lower available redox cofactors (NAD+, NADH, NADP+, and NADPH) than KP cells, as previously reported by Kottakis et al.19Kottakis F. Nicolay B.N. Roumane A. et al.LKB1 loss links serine metabolism to DNA methylation and tumorigenesis.Nature. 2016; 539: 390-395Crossref PubMed Scopus (205) Google Scholar In addition, the authors found that KP cells exhibited more robust growth than KL cells when grown in media without glutamine. This finding is particularly interesting because it is known that KP cells are reliant on glutamine,20Davidson S.M. Papagiannakopoulos T. Olenchock B.A. et al.Environment impacts the metabolic dependencies of Ras-driven non-small cell lung cancer.Cell Metab. 2016; 23: 517-528Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar suggesting that these KL cells have shifted their metabolic pathways to a more glutamine-dependent phenotype. That said, it must be recognized that evaluating metabolism in vitro, as done in this experiment, has significant limitations. Specifically, Davidson et al.20Davidson S.M. Papagiannakopoulos T. Olenchock B.A. et al.Environment impacts the metabolic dependencies of Ras-driven non-small cell lung cancer.Cell Metab. 2016; 23: 517-528Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar previously revealed that although KP cells in vitro were glutamine-dependent, KP tumors in vivo were not reliant on glutamine. Next, Zhang et al.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar evaluated whether the low oxygen consumption observed in vitro for KL cells was also observed in human KL tumors by leveraging a publicly available proteogenomic data set of human LUAD that included both KL and KP subsets. In this data set, differences were detected in the proteins involved in the oxidative phosphorylation (OXPHOS) pathway between KL and KP tumors, adding strength to their in vitro data. Whereas this data set did not directly address whether glutamine is used preferentially in KP versus KL tumors, Galan-Cobo et al.21Galan-Cobo A. Sitthideatphaiboon P. Qu X. et al.LKB1 and KEAP1/NRF2 pathways cooperatively promote metabolic reprogramming with enhanced glutamine dependence in KRAS-mutant lung adenocarcinoma.Cancer Res. 2019; 79: 3251-3267Crossref PubMed Scopus (160) Google Scholar reported that KRAS and KL mutant cells with inactivation in KEAP1, which hyperactivates NRF2, cause a dependency on glutaminolysis.21Galan-Cobo A. Sitthideatphaiboon P. Qu X. et al.LKB1 and KEAP1/NRF2 pathways cooperatively promote metabolic reprogramming with enhanced glutamine dependence in KRAS-mutant lung adenocarcinoma.Cancer Res. 2019; 79: 3251-3267Crossref PubMed Scopus (160) Google Scholar,22Romero R. Sayin V.I. Davidson S.M. et al.Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.Nat Med. 2017; 23: 1362-1368Crossref PubMed Scopus (367) Google Scholar Interestingly, a recent preprint by Härkönen et al.23Härkönen J. Pölönen P. Deen A.J. et al.Pan-cancer scale screening reveals NRF2 associated immunoevasive characteristics in non-small cell lung cancer and squamous malignancies. bioRxiv.https://www.biorxiv.org/content/10.1101/2022.05.15.489654v1.article-infoDate accessed: March 27, 2023Google Scholar revealed that activation of the NRF2 pathway strongly induces NAMPT, a rate-limiting enzyme in the biosynthesis of NAD. As KL cells have lower NAD and NADPH cofactors versus KP cells, this might offer a clue as to how mutations in KEAP1 can rescue oxidative metabolism in KL tumors. Future studies should evaluate how the metabolic profiles of KRAS, LKB1, and KEAP1 (KLK) or KRAS, TP53, and KEAP1 (KPK) mutant cells compared with the KL cells described by Zhang et al.,17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar as both KLK and KPK cell types have a similar dependency on glutamine. The authors then performed RNA-sequencing to dissect the expression levels of the glycolytic enzymes, aldolase, enolase, lactate dehydrogenase A, and glyceraldehyde-3-phosphate dehydrogenase in KL and KP cells. Because KL cells exhibited increased expression of these enzymes versus KP cells, the authors investigated the effect of HDAC6 inhibition on cell growth. As expected from the RNA-sequencing data, KL cells had decreased tumor size after HDAC6 inhibition in spheroid culture or subcutaneously in C57BL/6 mice.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar These results suggest that KL cells are sensitive to HDAC6 inhibition both in vitro and in vivo. More importantly, these findings are consistent with a recent study in triple-negative breast cancer, which revealed that HDAC6 is a regulator of glycolytic metabolism through protein degradation.24Dowling C.M. Hollinshead K.E.R. Di Grande A. et al.Multiple screening approaches reveal HDAC6 as a novel regulator of glycolytic metabolism in triple-negative breast cancer.Sci Adv. 2021; 7eabc4897Crossref PubMed Scopus (24) Google Scholar Inhibition of HDAC6 has also been found to help overcome proteasome inhibitor resistance in multiple myeloma,25Hideshima T. Qi J. Paranal R.M. et al.Discovery of selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma.Proc Natl Acad Sci U S A. 2016; 113: 13162-13167Crossref PubMed Scopus (94) Google Scholar which has led to a trial evaluating the HDAC6 inhibitor ACY1215 in combination with Pomalidomide/dexamethasone to treat relapsed/refractory multiple myeloma (NCT01997840). Human LUAD with KRAS and LKB1 co-mutations have reduced levels of OXPHOS compared with LUAD with KRAS/TP53 mutations.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar This has been recently corroborated in vivo by means of KRAS-driven murine lung cancer models from our laboratory.26Han M. Bushong E.A. Segawa M. et al.Spatial mapping of mitochondrial networks and bioenergetics in lung cancer.Nature. 2023; 615: 712-719Crossref Scopus (4) Google Scholar To evaluate the impact of HDAC6 inhibition on metabolism in KL and KP cell lines, the authors metabolically profiled the KL and KP cells after treatment with ACY1215. ACY1215 treatment did not change oxygen consumption rates in KL or KP cells, but only KP cells adapted their metabolism by increasing glucose/glutamine uptake after treatment. In addition, only KL cells exhibited a decrease in all glutathione levels (oxidized, reduced, and total) suggesting that KL cells are more sensitive to oxidative stress versus KP cells. Further metabolite profiling by liquid chromatography-mass spectrometry after HDAC6 inhibition in both KL and KP cells identified distinct metabolite changes. In both KL and KP cells, glucose-6-phosphate was reduced after treatment, confirming decreased glycolysis. That said, KP cells were able to increase carnitine and acyl-carnitine metabolites suggesting increased reliance on fatty-acid oxidation, which was not the case for KL cells.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar This metabolite profiling suggests a lower potential for metabolic adaptation in KL cells versus KP cells after HDAC6 treatment, which indicates that LKB1 mutational status impacts the ability of tumor cells to maintain energy homeostasis after HDAC6 inhibition. In addition, it further identified potential strategies for effective combinatorial treatment, by inhibiting key metabolism pathways including glutaminolysis, fatty-acid oxidation, and OXPHOS. After characterizing the metabolic impact of ACY1215 on KL and KP cells, the authors explored the impact of combining HDAC6 inhibition with glutaminase inhibition, a key enzyme in the glutaminolysis pathway. Previous work by our laboratory and others has revealed that combining the glutaminase inhibitor CB-839 with a glycolytic inhibitor is viable.15Momcilovic M. Bailey S.T. Lee J.T. et al.Targeted inhibition of EGFR and glutaminase induces metabolic crisis in EGFR mutant lung cancer.Cell Rep. 2017; 18: 601-610Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar,16Momcilovic M. Bailey S.T. Lee J.T. et al.The GSK3 signaling axis regulates adaptive glutamine metabolism in lung squamous cell carcinoma.Cancer Cell. 2018; 33: 905-921 e5Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar CB-839 has also been tested on several NSCLC trials, including in combination with osimertinib (NCT03831932) and the mTOR inhibitor MLN0128 (NCT04250545).27Riess J.W. Frankel P. Shackelford D. et al.Phase 1 trial of MLN0128 (sapanisertib) and CB-839 HCl (telaglenastat) in patients with advanced NSCLC (NCI 10327): rationale and study design.Clin Lung Cancer. 2021; 22: 67-70Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Whereas the results from these trials are not available, other trials evaluating CB-839 have been disappointing. For example, KEAPSAKE (NCT04265534), a clinical trial in KEAP1 or NRF2 mutant NSCLC that combined CB-839 with frontline chemoimmunotherapy was terminated early owing to a lack of benefit. Mutant KEAP1 or NRF2 NSCLC was targeted on KEAPSAKE because these genes are key modulators of oxidative stress, such that the inhibition of glutaminase by means of CB-839 was expected to have a major effect on oxidative stress and tumor response. In the current study, the combination of HDAC6 inhibitor and CB-839 both in vitro and in vivo was more effective in KL than KP cells, suggesting that LKB1 mutational status plays an important role in oxidative stress response and results in increased sensitivity to combinatorial HDAC6 and glutaminase inhibition. In summary, the study of Zhang et al.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar supports further exploration of HDAC6 inhibition in KRAS and LKB1 co-mutant NSCLC.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar The previous failure of the KEAPSAKE trial, which similarly hypothesized that targeting glutaminolysis and oxidative stress by means of glutaminase inhibition would be beneficial to patients with KEAP1/NRF2 mutations, emphasizes the importance of the following: (1) precise patient stratification for treatments that target oxidative stress and metabolism; (2) targeting multiple metabolic nodes; and (3) identifying the metabolic nodes most vulnerable to perturbation. In this context, it is important to identify the patient population that is most likely to benefit from ACY1215 plus or minus CB-839 and, to do so, the role of KEAP1 and NRF2 co-mutations in both KL and KP tumors must be addressed as KPK and KLK cells have been reported to have increased sensitivity to glutaminase inhibition with CB-839.21Galan-Cobo A. Sitthideatphaiboon P. Qu X. et al.LKB1 and KEAP1/NRF2 pathways cooperatively promote metabolic reprogramming with enhanced glutamine dependence in KRAS-mutant lung adenocarcinoma.Cancer Res. 2019; 79: 3251-3267Crossref PubMed Scopus (160) Google Scholar,22Romero R. Sayin V.I. Davidson S.M. et al.Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.Nat Med. 2017; 23: 1362-1368Crossref PubMed Scopus (367) Google Scholar Additional questions arise in the context of an ACY1215 with or without CB-839 addition to frontline chemoimmunotherapy. Previous work has found that HDAC6 inhibition has immunomodulatory effects in human tumors and KP and EGFR mouse models,28Adeegbe D.O. Liu Y. Lizotte P.H. et al.Synergistic immunostimulatory effects and therapeutic benefit of combined histone deacetylase and bromodomain inhibition in non-small cell lung cancer.Cancer Discov. 2017; 7: 852-867Crossref PubMed Scopus (112) Google Scholar but they did not use KL mouse models. Therefore, it would be important to understand what effect HDAC6 inhibition has on the immune composition of KL versus KP tumors, something that was not explored by Zhang et al.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar Furthermore, would adding ACY1215 or CB-839 to a chemoimmunotherapy regimen improve responses in tumors with dysregulated LKB1, KEAP1, and NRF2? Disappointing results from the KEAPSAKE trial may suggest otherwise; nevertheless, follow-up preclinical studies evaluating the impact of HDAC6 inhibition on the remodeling of the tumor immune microenvironment are warranted. Ultimately, Zhang et al.17Zhang H. Nabel C.S. Li D. et al.Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant KRAS driven non-small cell lung cancer.J Thorac Oncol. 2023; 18: 881-894Abstract Full Text Full Text PDF Scopus (2) Google Scholar should be praised for adding to our understanding of the metabolic dependencies in KRAS-mutant LUAD, especially those that harbor LKB1 co-mutations, as this work moves the field closer to rationally designed therapeutic approaches that are desperately needed to improve outcomes in this difficult-to-treat patient population. Brandon Nadres: Writing – original draft preparation. Milica Momcilovic, David B. Shackelford, Aaron Lisberg: Writing – reviewing and editing. Histone Deacetylase 6 Inhibition Exploits Selective Metabolic Vulnerabilities in LKB1 Mutant, KRAS Driven NSCLCJournal of Thoracic OncologyVol. 18Issue 7PreviewIn KRAS-mutant NSCLC, co-occurring alterations in LKB1 confer a negative prognosis compared with other mutations such as TP53. LKB1 is a tumor suppressor that coordinates several signaling pathways in response to energetic stress. Our recent work on pharmacologic and genetic inhibition of histone deacetylase 6 (HDAC6) revealed the impaired activity of numerous enzymes involved in glycolysis. On the basis of these previous findings, we explored the therapeutic window for HDAC6 inhibition in metabolically-active KRAS-mutant lung tumors. Full-Text PDF Open Access