A coding single-nucleotide polymorphism in lysine demethylase KDM4A associates with increased sensitivity to mTOR inhibitors

Abstract

SNPs occur within chromatin-modulating factors; however, little is known about how these variants within the coding sequence affect cancer progression or treatment. Therefore, there is a need to establish their biochemical and/or molecular contribution, their use in subclassifying patients, and their impact on therapeutic response. In this report, we demonstrate that coding SNP-A482 within the lysine tridemethylase gene KDM4A/JMJD2A has different allelic frequencies across ethnic populations, associates with differential outcome in patients with non-small cell lung cancer (NSCLC), and promotes KDM4A protein turnover. Using an unbiased drug screen against 87 preclinical and clinical compounds, we demonstrate that homozygous SNP-A482 cells have increased mTOR inhibitor sensitivity. mTOR inhibitors significantly reduce SNP-A482 protein levels, which parallels the increased drug sensitivity observed with KDM4A depletion. Our data emphasize the importance of using variant status as candidate biomarkers and highlight the importance of studying SNPs in chromatin modifiers to achieve better targeted therapy.

Significance: This report documents the first coding SNP within a lysine demethylase that associates with worse outcome in patients with NSCLC. We demonstrate that this coding SNP alters the protein turnover and associates with increased mTOR inhibitor sensitivity, which identifies a candidate biomarker for mTOR inhibitor therapy and a therapeutic target for combination therapy.

Figure 1. KDM4A SNP-A482 (rs586339) correlates with worse outcome in NSCLC patients

(A) Schematic of the human KDM4A protein is shown with both the protein domains and the position of the coding SNP rs586339 (E482A). Jumonji (JmjN and JmjC), PHD and Tudor (T) domains are represented. (B) E482 is the conserved allele. The alignment of sequence surrounding E482A is shown for multiple species. (C) HapMap frequencies for rs586339 are presented (August 2010 HapMap public release #28) (13). ASW- African Ancestry in SW USA (n=57); CEU- U.S. Utah residents with ancestry from northern and western Europe (n=113); CHB- Han Chinese in Beijing, China (n=135); CHD- Chinese in Metropolitan Denver, CO, USA (n=109); GIH- Gujarati Indians in Houston, TX, USA (n=99); JPT- Japanese in Tokyo, Japan (n=113); LWK- Luhya in Webuye, Kenya (n=110); MKK- Maasai in Kinyawa, Kenya (n=155); MXL- Mexican Ancestry in Los Angeles, CA, USA (n=58); TSI- Toscani in Italia (n=102); YRI- Yoruba in Ibadan, Nigeria (n=147). (D) Representative KDM4A sequencing plots from three different lung cancer cell lines- homozygote wild type (WT, GAA:GAA), heterozygote (HET, GAA:GCA) and homozygote SNP (A-482, GCA:GCA). (E) KDM4A SNP-A482 stratification for late stage NSCLC patients. HR represents the hazard ratio, 95%CI the 95% confidence interval, p the p value, n the number of patients in each category, AA the genotype for homozygote wild-type (E482), AC for heterozygote, CC for homozygote SNP (A482). Gray boxes highlight the parameters with significance, including the data in panels B-F. Frequency comparisons were tested using the chi-square test, and Hazard Ratios were calculated using a Cox model. Also see supplementary Figure S1.

Figure 2. SNP-A482 promotes KDM4A ubiquitination and turnover

(A) KDM4A WT and SNP-A482 comparably demethylate H3K36me3 and H3K9me3. 3xHA-KDM4A WT and 3xHA-KDM4A SNP-A482 were transfected into RPE cells, fixed and stained with the indicated antibodies. The graph represents an average of two experiments. At least fifty cells were scored per experiment. (B) KDM4A SNP-A482 exhibits a two-fold greater ubiquitination than KDM4A WT. GFP-KDM4A WT and GFP-KDM4A SNP-A482 were transfected into HEK 293T cells prior to immunoprecipitation with a GFP antibody under denaturing conditions and immunoblotted with the indicated antibodies. Quantification was performed with ImageJ. The graph represents an average of five independent experiments that show the ratio of ubiquitin signal to the amount of immunoprecipitated protein. (C) GFP- A482 co-immunoprecipitates more MYC-Cullin1 than GFP-WT KDM4A. GFP-WT and GFP- A482 were transfected into HEK 293T cells before being immunoprecipitated with a GFP antibody and immunoblotted with the indicated antibodies. (D) GFP- A482 has a shorter half-life than GFP-WT KDM4A. HEK 293T cells overexpressing GFP-WT or GFP-A482 were treated with cycloheximide and western blotted. The y axis represents the ratio of GFP-tagged KDM4A relative to time 0, which was normalized to β-actin. The average of 16 independent experiments is shown. All error bars represent the SEM. P values were determined by a two-tailed student’s t test; * represents p<0.05.

Figure 3. KDM4A SNP-A482 impacts cellular sensitivity to specific drugs

(A) Volcano plot representing statistical significance (inverted Y axis) versus the effect of KDM4A SNP-A482 on drug sensitivity. Compounds above the X axis are statistically significant (p<0.05). Eighty seven cell lines and eighty eight compounds were used; the statistical significance of the enrichment for mTOR inhibitors in the group of drugs linked to the SNP status is indicated (p=0.002). (B) A list of compounds with statistically different sensitivity in (A), their associated targets and corresponding p values are shown. p values were calculated with Fisher’s exact test. Also see supplementary Figure S2 and supplementary Table 1.

Figure 4. KDM4A levels impact cellular sensitivity to mTOR inhibitors

(A) KDM4A protein levels decrease upon Rapamycin treatment. HEK 293T cells were treated with 100ng/ml of Rapamycin for 24h. (B) Graphical representation of an average of three independent experiments from (A). (C) KDM4A RNA levels are stable upon Rapamycin treatment. HEK 293T cells were treated with 100ng/ml of Rapamycin for 24h before RNA was harvested. An average of three independent quantitative RT-PCR experiments is represented. (D-E) Endogenous KDM4A SNP-A482 protein levels decrease more upon Rapamycin treatment than WT KDM4A. Lung cell lines homozygous for KDM4A SNP-A482 (D; H290 and RERF-LC-KJ) or WT (E; LU99B and H2591) were treated with 100ng/ml of Rapamycin for 24h. Independent replicates (r1 and r2) are shown per time point. Quantification was performed with ImageJ. The numbers under the blots represent the ratio of the amount of KDM4A to the amount of Actinin, normalized to vehicle. (F) HEK 293T cells transfected with three different shRNAs directed against KDM4A have increased sensitivity to Rapamycin when compared to control vector transfected cells. HEK 293T cells were seeded 24h after transfection and treated with 100ng/ml of Rapamycin 24h after seeding. The graphs represent the doubling time between 5h and 35h after Rapamycin treatment. An average of three independent experiments is represented. (G) HEK 293T cells transfected with shRNA 4A.6 are more sensitive to AZD8055 than cells transfected with the control vector. Cells were seeded 24h after the second shRNA transfection and were then treated with the indicated drugs and associated concentrations 24h later. 48h after treatment, samples were analyzed by MTT assay. The assays were normalized to a sample collected and assayed at the treatment time. The Y axis represents the viability ratio relative to DMSO. The average of three independent experiments is represented. (H) Model. KDM4A depletion of SNP-A482 enhanced the sensitivity to mTOR inhibitors. All error bars represent the SEM. p values were determined by a two-tailed student’s t test; * represents p<0.05. Also see supplementary Figure S3.