Histone Deacetylase 6 Inhibition Exploits Selective Metabolic Vulnerabilities in LKB1 Mutant, KRAS Driven NSCLC

Abstract

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.

Keywords: Glutaminase inhibition; Glycolysis; HDAC6; KRAS; LKB1; Oxidative stress.

Figure 1:. Evaluation of metabolic differences between KRAS/TP53 and KRAS/LKB1 comutant cells.

(A) Growth rate, indicated as doublings per day. (B) Glucose, glutamine consumption and lactate production in media conditioned during cell growth. (C) Growth rate under conditions of glucose and glutamine withdrawal. (D) Basal oxygen consumption rate. (E) NAD co-factor levels, normalized to cell counts and referenced to mean of KP condition. (F) Total, oxidized, and reduced glutathione levels, normalized to cell counts and referenced to mean of KP condition. For each figure, each data point represents the average of 2–3 biological replicates. Three distinct cell lines were used from each genotype, and the results of two representative experiments are shown. Statistical analysis was performed with unpaired, parametric student’s T-test. For all panels, P-values noted as follows: *P<0.05, **P<0.01****P<0.0001.

Figure 2:. KRAS/LKB1 models demonstrate enhanced sensitivity to HDAC6 inhibition.

(A) Dose response of a panel of KL and KP NSCLC murine cell lines to ACY1215 as measured by CCK8 cell death assay (n=3, mean +/− SEM) (IC50 Values; KLA 3.4μM, KLB 1.9μM, KLC 2.8μM, KPA 14.2μM, KPC 12.7μM, KPD 12.3μM). (B) Dose response of a panel of KL and KP NSCLC murine cell lines to BAS-2 as measured by CCK8 cell death assay (n=3, mean +/− SEM) (IC50 Values; KLA 33.2μM, KLB 23.1μM, KLC 30.6μM, KPA 48.2μM, KPC 59.1μM, KPD 41.2μM). (C) Images of KLA cells grown in Matrigel for 7 days following treatment with 1μM of ACY1215 or 10μM of BAS-2 (n=3). (D) Scatter dot plot shows the size of spheroids in all treatment groups (E) Images of KPA cells grown in Matrigel for 7 days following treatment with 1μM of ACY1215 or 10μM of BAS-2 (n=3). (F) Scatter dot plot shows the size of spheroids in all treatment groups. (G) NAD co-factor levels and (H) Glutathione levels in in subcutaneous tumors derived from implantation of KLA and KPA murine cancer cell lines. (I&J) C57BL/6 mice were subcutaneously inoculated with 1×10⁶ KL (I) or KP (J) cells and following tumor formation were treated with 50mg/kg of ACY1215 or vehicle control for 7 days. Tumors were measured and plotted as percentage change in tumor size following treatment. Statistical analysis was performed with unpaired, parametric student’s T-test. For all panels, P-values noted as follows: **P<0.01,***P<0.001.

Figure 3:. Metabolic alterations associated with pharmacologic HDAC6 inhibition

Cells were treated with ACY1215 1 uM or 0.01% DMSO Vehicle control for 24 hours before making the following metabolic assessments: (A) Glucose, glutamine consumption and lactate production. (B) Basal oxygen consumption rate. (C) NAD co-factor levels, normalized to cell counts and referenced to mean of KP condition. (D) Total, oxidized, and reduced glutathione levels, normalized to cell counts and referenced to mean of KP condition. (E) Variable importance projection of differential enrichment of metabolites measured by LC/MS in the KP background, comparing 24 hour ACY1215 1uM versus 0.01% DMSO control. (F) Same analysis in the KL background. (G) Venn diagram illustrating overlap of metabolites identified through VIP. (H) Relative metabolite levels for glucose-6-phosphate, 3-phosphoglycerate, phosphoenolpyruvate and citrate, as normalized to the vehicle-treated KP condition. For each figure, each data point represents the average of 2–3 biological replicates. Three distinct cell lines were used from each genotype, and the results of two representative experiments are shown. For panels A and B, statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons test. For panels C, D, and H, statistical analysis was performed with two-way ANOVA with Tukey’s multiple comparisons test. For all panels, P-values noted as follows: *P<0.05, **P<0.01,***P<0.001, ****P<0.0001.

Figure 4:. Enhanced efficacy of combining HDAC6 inhibitors with Glutaminase inhibitors in KRAS/LKB1 NSCLC modes.

(A&B) Dose response of KLB (A) and KPA (B) NSCLC murine cell lines to ACY1215, CB-839 and combinations as measured by CCK8 cell death assay (n=3, mean +/− SEM, drug concentration is Log₁₀). (C) Images of KLB cells grown in Matrigel for 7 days following treatment with 1μM of ACY1215, 50nM CB-839 or combination (1μM of ACY1215 & 50nM CB-839) (n=3). (D) Scatter dot plot shows the size of KLB spheroids in all treatment groups. (E) Images of KPA cells grown in Matrigel for 7 days following treatment with 1μM of ACY1215, 50nM CB-839 or combination (1μM of ACY1215 & 50nM CB-839) (n=3). (F) Scatter dot plot shows the size of KPA spheroids in all treatment groups. (G) C57BL/6 mice were subcutaneously inoculated with 1×10⁶ KL cells and following tumor formation were treated with vehicle, ACY1215 (50mg/kg), CB-839 (200mg/kg) or combination for 14 days. At the end of the experiment mice were culled and tumors volume was measured [(length × width²)/2]. Statistical analysis for 3D spheroids was performed with unpaired, parametric student’s T-test. Statistical analysis for mouse models was performed with one-way anova, Dunnett’s multiple comparisons. For all panels, P-values noted as follows: *P<0.05, **P<0.01,***P<0.001, ****P<0.0001.