Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies

Abstract

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention.

Keywords: Cancer; Hematology; Oncology.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Glutamine is an important contributor of carbon substrate to the TCA cycle.

The mass isotopomer distribution of the various TCA cycle intermediates after incubation of MM1S and RPMI-8226 human myeloma cell lines (HMCLs) in cell culture media containing either U[13C5]Glutamine or U[13C6]Glucose. The (m+4) or (m+5) isotopomers highlighted in green circles represent the isotopomer derived from U[13C5]Glutamine, and the (m+2) isotopomers highlighted in red circles are derived from U[13C6]Glucose. The unlabeled TCA cycle intermediates represented by (m+0) are highlighted in black circles. Bar graph values represent the averages of the individual data points (triplicates) that overlay the bar graph. The error bars represent ± SEM. Comparisons were made using the 2-tailed Student t test, and significance was defined as P < 0.05.

Figure 2. Glucose is an important contributor of carbon substrate for the formation of lactate.

The mass isotopomer distribution of lactate after incubation of MM1S or RPMI-8226 human myeloma cell lines (HMCLs) in cell culture media containing either U[13C5]Glutamine or U[13C6]Glucose. The (m+3) isotopomers highlighted in green and red circles represent the isotopomer derived from U[13C5]Glutamine and U[13C6]Glucose, respectively. Bar graph values represent the averages of the individual data points (triplicates) that overlay the bar graph. The error bars represent ± SEM. Comparisons were made using the 2-tailed Student t test, and significance was defined as P < 0.05.

Figure 3. 2-HG is detectable in human myeloma cell lines.

Intracellular concentrations of 2-hydroxyglutarate (2-HG) measured by gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) methods in each of 5 different human myeloma cell lines (HMCLs).

Figure 4. Glutamine is an important contributor of carbon substrate for the formation of 2-HG.

The mass isotopomer distribution of 2-hydroxyglutarate (2-HG) after incubation of MM1S or RPMI-8226 human myeloma cell lines (HMCLs) in cell culture media containing either U[13C5]Glutamine or U[13C6]Glucose. The (m+5) isotopomers highlighted in green circles represent the isotopomer derived from U[13C5]Glutamine, and the (m+2) isotopomers highlighted in red circles are derived from U[13C6]Glucose. Bar graph values represent the averages of the individual data points (triplicates) that overlay the bar graph. The error bars represent ± SEM. Comparisons were made using the 2-tailed Student t test, and significance was defined as P < 0.05.

Figure 5. Glutamine uptake is higher in BM plasma cells compared with the remainder of the BM mononuclear cells.

Mass isotopomer distribution of glutamate representing the results of ex vivo glutamine anaplerosis of U[13C5]Glutamine into CD138+ and CD138– cells derived from the BM aspirates of patients with multiple myeloma (MM) (n = 4). Bar graph values represent the averages of the individual data points that overlay the bar graph. The error bars represent ± SEM. Comparisons were made using the 2-tailed Student t test, and significance was defined as P < 0.05.

Figure 6. 2-HG differentiates between monoclonal gammopathy of undetermined significance and multiple myeloma.

(A) Dot plot graph depicting the individual (blue circles and red squares), mean (red and blue line), and ± SEM (black error bars) of 2-hydroxyglutarate (2-HG) concentrations in the BM supernatant of patients in the monoclonal gammopathy of undetermined significance (MGUS) (n = 5) and multiple myeloma (MM) (n = 15) cohorts. Comparisons were made by the Mann-Whitney test, and significance was defined as P < 0.05. (B) Bar graph representing the percentage of monoclonal gammopathy of undetermined significance (MGUS) (n = 5) and multiple myeloma (MM) (n = 15) patients with 2-hydroxyglutarate (2-HG) levels in the BM supernatant higher than the median 2-HG level of the cohort. Comparisons were made by the 2-tailed Fisher’s exact test, and significance was defined as P < 0.05.

Figure 7. 2-HG levels in the BM supernatant predicts for shorter time to progression of smoldering multiple myeloma to multiple myeloma.

(A) Kaplan-Meier curve showing the time to progression (TTP) of patients with smoldering multiple myeloma (SMM) with a 2-hydroxyglutarate (2-HG) level in the BM supernatant higher than the median 2-HG level (elevated 2-HG) (n = 10) compared with patients with SM) with a 2-HG level in the BM supernatant lower or equal to the median 2-HG levels of the cohort (nonelevated 2-HG) (n = 15). Comparisons were made by the Wilcoxon test, and significance was defined as P < 0.05. (B) Bar graph representing the percentage of SMM patients with 2-HG levels in the BM supernatant higher than the median 2-HG level (elevated 2-HG) (n = 10) or lower than the median 2-HG level (nonelevated 2-HG) (n = 15) who progressed to MM in 1 year from the time of 2-HG level assessment. Comparisons were made by the 2-tailed Fisher’s exact test, and significance was defined as P < 0.05. (C) Dot plot graph depicting the individual (red circles and blue squares), mean (red and blue line), and ± SEM (black error bars) of 2-HG concentrations in the BM supernatant of patients with SMM who progressed to MM within 12 months or less (n = 13) and later than 12 months (n = 12). Comparisons were made by the Mann-Whitney test, and significance was defined as P < 0.05.

Figure 8. c-Myc expression in BM plasma cells correlates with 2-HG levels in the BM supernatant.

(A) Dot plot graph depicting the individual (red circles and blue squares), mean (red and blue line), and ± SEM (black error bars) of 2-hydroxyglutarate (2-HG) concentrations in the BM supernatant of patients with smoldering multiple myeloma (SMM) whose BM biopsies show either ≥ 20% (n = 14) or < 20% (n = 11) clonal plasma cells with c-MYC nuclear staining. Comparisons were made by the Mann-Whitney test, and significance was defined as P < 0.05. (B) Example of a BM biopsy (magnification 40×) showing ≥ 20% clonal plasma cells with c-MYC nuclear staining (brown). (C) Example of a BM biopsy (magnification 40×) showing <20% clonal plasma cells with c-MYC nuclear staining (brown).

Figure 9. 2-HG levels in the peripheral blood predicts for shorter time to progression of smoldering multiple myeloma to multiple myeloma.

(A) Kaplan-Meier curve showing the time to progression (TTP) of patients with smoldering multiple myeloma (SMM) with a 2-hydroxyglutarate (2-HG) level in the peripheral blood plasma higher than the median 2-HG level (elevated 2-HG) (n = 14) compared with patients with SMM with a 2-HG level in the peripheral blood plasma lower or equal to the median 2-HG levels of the cohort (nonelevated 2-HG) (n = 11). Comparisons were made by the Wilcoxon test, and significance was defined as P < 0.05. (B) Bar graph representing the percentage of SMM patients with 2-HG levels in the peripheral blood plasma higher than the median 2-HG level (elevated 2-HG) (n = 14) or lower than the median 2-HG level (nonelevated 2-HG) (n = 11) that progressed to MM in 1 year from the time of 2-HG level assessment. Comparisons were made by the 2-tailed Fisher’s exact test, and significance was defined as P < 0.05. (C) Dot plot graph depicting the individual (red circles and blue squares), mean (red and blue line), and ± SEM (black error bars) of 2-HG concentrations in the peripheral blood plasma of patients with SMM who progressed to MM within 12 months or less (n = 13) and later than 12 months (n = 12). Comparisons were made by the Mann-Whitney test, and significance was defined as P < 0.05.