Shining a light on metabolic vulnerabilities in non-small cell lung cancer.

Metabolic reprogramming is a hallmark of cancer which contributes to essentialprocesses required for cell survival, growth, and proliferation. Non-small cell lung cancer(NSCLC) is the most common type of lung cancer and its genomic classification has given riseto the design of therapies targeting tumors harboring specific gene alterations that causeaberrant signaling. Lung tumors are characterized with having high glucose and lactate use,and high heterogeneity in their metabolic pathways. Here we review how NSCLC cells withdistinct mutations reprogram their metabolic pathways and highlight the potential metabolicvulnerabilities that might lead to the development of novel therapeutic strategies.</div

Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant, KRAS driven NSCLC

Introduction: In 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. Methods: Using cell lines derived from mouse autochthonous tumors bearing the KRAS/LKB1 (KL) and KRAS/TP53 mutant genotypes to control for confounding germline and somatic mutations in human models, we characterize the metabolic phenotypes at baseline and in response to HDAC6 inhibition. The impact of HDAC6 inhibition was measured on cancer cell growth in vitro and on tumor growth in vivo. Results: Surprisingly, KL-mutant cells revealed reduced levels of redox-sensitive cofactors at baseline. This is associated with increased sensitivity to pharmacologic HDAC6 inhibition with ACY-1215 and blunted ability to increase compensatory metabolism and buffer oxidative stress. Seeking synergistic metabolic combination treatments, we found enhanced cell killing and antitumor efficacy with glutaminase inhibition in KL lung cancer models in vitro and in vivo. Conclusions: Exploring the differential metabolism of KL and KRAS/TP53-mutant NSCLC, we identified decreased metabolic reserve in KL-mutant tumors. HDAC6 inhibition exploited a therapeutic window in KL NSCLC on the basis of a diminished ability to compensate for impaired glycolysis, nominating a novel strategy for the treatment of KRAS-mutant NSCLC with co-occurring LKB1 mutations.</p

Histone deacetylase 6 inhibition exploits selective metabolic vulnerabilities in LKB1 mutant, KRAS driven NSCLC

Introduction: In 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. Methods: Using cell lines derived from mouse autochthonous tumors bearing the KRAS/LKB1 (KL) and KRAS/TP53 mutant genotypes to control for confounding germline and somatic mutations in human models, we characterize the metabolic phenotypes at baseline and in response to HDAC6 inhibition. The impact of HDAC6 inhibition was measured on cancer cell growth in vitro and on tumor growth in vivo. Results: Surprisingly, KL-mutant cells revealed reduced levels of redox-sensitive cofactors at baseline. This is associated with increased sensitivity to pharmacologic HDAC6 inhibition with ACY-1215 and blunted ability to increase compensatory metabolism and buffer oxidative stress. Seeking synergistic metabolic combination treatments, we found enhanced cell killing and antitumor efficacy with glutaminase inhibition in KL lung cancer models in vitro and in vivo. Conclusions: Exploring the differential metabolism of KL and KRAS/TP53-mutant NSCLC, we identified decreased metabolic reserve in KL-mutant tumors. HDAC6 inhibition exploited a therapeutic window in KL NSCLC on the basis of a diminished ability to compensate for impaired glycolysis, nominating a novel strategy for the treatment of KRAS-mutant NSCLC with co-occurring LKB1 mutations.</p

Continuous activation of ER-K-ras^G12D by 500 µg tamoxifen treatment induced lung hyperplasia and adenoma formation in P53 L/L, LSL-ER-K-ras mice.

<p>A) Continuous activation of ER-K-ras^G12D by 500 µg tamoxifen I.P. daily injection for 12 weeks is sufficient to drive lung adenoma formation in P53 L/L, LSL-ER-K-ras mice after adeno-Cre treatment. The adenoma were positive for Ki67 immunostaining. B) The incidence of lung hyperplasia and/or adenoma formation in <i>P53L/L</i>, <i>LSL-ER-K-ras^G12D</i> mice was shown after adeno-Cre treatment in the absence or presence of tamoxifen treatment.</p

<i>RMRP</i> depletion leads to reduced levels of <i>RMRP</i> transcript.

<p>Total RNA was produced from E13.5 MEFs and <i>RMRP</i> level was measured by A. qRT-PCR B. Northern blot using either a sense or antisense <i>RMRP</i> probe. Error bars represent SD of three replicas.</p

Targeting of murine <i>RMRP</i>.

<p>A. Murine targeting vector (MTV) B. Southern blot of ES cells following selection for alleles with integrated MTV (the southern probe is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026270#pone-0026270-g001" target="_blank">figure 1a</a>) C. PCR analysis of RC (<i>RMRP</i> conditional) pups D. PCR analysis of pups derived from the interbreeding of RC mice and mice expressing CMV-Cre.</p

<i>RMRP</i> depletion is embryonic lethal.

<p><i>RMRP</i> depletion is embryonic lethal.</p

Sustained activation of ER-K-ras^G12D induced by tamoxifen treatment is important for cell proliferation, anchorage-independent cell growth, cell invasiveness and tumor maintenance.

<p>A) Cell number counting of indicated MEFs after withdrawal of tamoxifen after 6 passages in the presence of tamoxifen. The PEK-C MEFs always without tamoxifen treatment and the PEK-C MEFs always kept in tamoxifen were used as control. B) Tamoxifen were withdrawn from the PEK-C MEFs at day 5, 10 and 28 after continuous tamoxifen treatment in soft agar. C) Tamoxifen withdrawal from the PEK-C MEFs after indicated days of tamoxifen treatment in cell invasiveness assay in matrigel. D) Tamoxifen withdrawal for indicated time in the <i>P53L/L</i>, <i>LSL-ER-K-ras^G12D</i> mice previously treated for 12 weeks of tamoxifen. The typical lung pathology and cleaved caspase-3 immunostaining were shown.</p

Genes near <i>RMRP</i> are not essential for cellular viability.

<p>MEFs from E13.5 mice of either A. WT or B. <i>RMRP</i>+/− were transfected with siRNAs targeting <i>Ccdc107</i> or <i>E130</i>. Three days later RNA was extracted from the cells and qRT-PCR was preformed using primers for <i>RMRP</i>, <i>Ccdc107</i> or <i>E130</i>. C. The same cells as in A and B were plated (5000 cells/well) in a 96 well plate and 7 days post transfection cell number was assessed by Cell titer glow. Error bars represent SD of three replicas.</p

Treatment of tamoxifen at an optimal dose induced the activation of ER-K- ras^G12D and downstream signaling.

<p>A) The <i>P53−/−</i>, <i>ER-K-ras^G12D</i> MEFs (PEK-C) derived from male embryo was counted for cell number after indicated dosage of tamoxifen treatment after 5 passages. B) Relative expression level of <i>K-ras</i> in indicated MEFs with or without tamoxifen treatment. PEK: <i>P53L/L</i>, <i>Loxp-Stop-Loxp ER-K-ras^G12D</i>; PEK-C: <i>P53−/−</i>, <i>ER-K-ras^G12D</i>; PK: <i>P53L/L</i>, <i>Loxp-Stop-Loxp-K- ras^G12D</i>; PKC: <i>P53−/−</i>, <i>K- ras^G12D</i>. (C) Detection of the ER-K-ras^G12D expression in PEK-C cells with or without 0.05 µM tamoxifen treatment. D) Detection of K-ras and ER-K-ras protein level in MEFs infected with or without adeno-Cre in the presence or absence of 0.05 µM tamoxifen treatment in indicated MEFs. β-actin serves as internal control. E) The activation of Ras effector PI3K pathway signaling was confirmed by western blot.</p