In vivo assessment of glutamine anaplerosis into the TCA cycle in human pre-malignant and malignant clonal plasma cells

Abstract

Background: Overexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown.

Methods: Human volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labeled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma.

Results: Despite notable heterogeneity, stable isotope-resolved metabolomics (SIRM) revealed that the mean 13C-labeled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients.

Conclusion: Measurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM.

Trial registration: NCT03384108 and NCT03119883.

Keywords: Glutamine; Myeloma; Plasma cell malignancies; Stable isotope metabolomics.

Conflict of interest statement

There are no competing interests or conflicts of interest among any of the authors.

Figures

Fig. 1

a Timeline of procedures/evaluations performed on the volunteers during the study day. b Plasma glutamine enrichment during the 5-13C-glutamine infusion in each of the volunteers (N = 7). c Glutamine enrichment in paired bone marrow and peripheral blood plasma after 60 min of the 5-13C-glutamine infusion from each of the volunteers (N = 7). d Comparison of mean relative 13C fractional enrichment of TCA cycle intermediates in CD138+ and CD138- cells from the bone marrows of volunteers (N = 3). Error bars represent SEM. n.s. is non-significant by paired t test

Fig. 2

a Glutamine enrichment in bone marrow plasma after 60 min of the [13C5]-glutamine infusion in patients with MGUS (N = 11) and MM (N = 12). Error bars represent SEM. n.s. is non-significant by unpaired t test. b Comparison of mean relative 13C fractional enrichment of TCA cycle intermediates in CD138+ and CD138- cells from the bone marrows of MGUS (N = 10) and MM (N = 11). Error bars represent SEM. n.s. is non-significant, *p < 0.05, **p < 0.01, and ***p < 0.001 by paired t test. c Relative 13C fractional enrichment (CD138+/CD138-) of TCA cycle intermediates from each MGUS (N = 10) and MM (N = 11) patient.

Fig. 3

a Hierarchical clustering analysis on the overall differential expression of genes between MGUS (N = 6) and MM (N = 10) patients. b Relative differences in mRNA expression of c-Myc, GLS, ASCT2, and SN2 between CD138+ cells from MGUS (N = 6) and MM (N = 10) patients in context of their effect on glutamine transport into the cell

Fig. 4

a Violin plots comparing the median concentrations of different TCA metabolites in the bone marrow plasma between MGUS (N = 11) and MM (N = 12) groups. Data was analyzed by Mann-Whitney U test where ***p < 0.001, **p < 0.01, *p < 0.05, #p < 0.1 but ≥ 0.05, n.s. non-significant. b Violin plots comparing the median concentrations of different TCA metabolites in the peripheral blood plasma between MGUS (N = 11) and MM (N = 12) groups. Data was analyzed by Mann-Whitney U test where ***p < 0.001, **p < 0.01, *p < 0.05, #p < 0.1 but ≥ 0.05, n.s. non-significant. c Violin plots comparing the median concentrations of aspartate and glutamate in the bone marrow plasma between the volunteers (N = 7), MGUS (N = 11), and MM (N = 12) groups. Data was analyzed by Mann-Whitney U test where ***p < 0.001, **p < 0.01, *p < 0.05, #p < 0.1 but ≥ 0.05, n.s. non-significant. d XY-correlation plots comparing the concentrations of aspartate and glutamate in the bone marrow plasma of patients with MGUS and MM and the percentage of clonal plasma cells in their bone marrow

Fig. 5

a Immunohistochemical staining of the bone marrow for CD138+ clonal plasma cells in MM patient #2 with primary plasma cell leukemia as well as peripheral smear evaluation demonstrating the presence of circulating plasma cells. b Positron emission tomography-computed tomography (PET/CT) demonstrating the presence of a large plasmacytoma in the right chest wall as well as immunohistochemical staining of the bone marrow for CD138+ clonal plasma cells in MM patient #7 with no marrow involvement. c Graphical visualization of the concentrations of the TCA cycle intermediate such as glutamate, succinate, fumarate, malate, and aspartate in the bone marrow plasma of MM patient #2 with primary plasma cell leukemia and MM patient #7 with respect to that of the remainder of the MM patients (represented by the mean (dashed line) and maximum and minimum (dotted lines) values)