Metabolic Changes Are Associated with Melphalan Resistance in Multiple Myeloma

Abstract

Multiple myeloma is an incurable hematological malignancy that impacts tens of thousands of people every year in the United States. Treatment for eligible patients involves induction, consolidation with stem cell rescue, and maintenance. High-dose therapy with a DNA alkylating agent, melphalan, remains the primary drug for consolidation therapy in conjunction with autologous stem-cell transplantation; as such, melphalan resistance remains a relevant clinical challenge. Here, we describe a proteometabolomic approach to examine mechanisms of acquired melphalan resistance in two cell line models. Drug metabolism, steady-state metabolomics, activity-based protein profiling (ABPP, data available at PRIDE: PXD019725), acute-treatment metabolomics, and western blot analyses have allowed us to further elucidate metabolic processes associated with melphalan resistance. Proteometabolomic data indicate that drug-resistant cells have higher levels of pentose phosphate pathway metabolites. Purine, pyrimidine, and glutathione metabolisms were commonly altered, and cell-line-specific changes in metabolite levels were observed, which could be linked to the differences in steady-state metabolism of naïve cells. Inhibition of selected enzymes in purine synthesis and pentose phosphate pathways was evaluated to determine their potential to improve melphalan's efficacy. The clinical relevance of these proteometabolomic leads was confirmed by comparison of tumor cell transcriptomes from newly diagnosed MM patients and patients with relapsed disease after treatment with high-dose melphalan and autologous stem-cell transplantation. The observation of common and cell-line-specific changes in metabolite levels suggests that omic approaches will be needed to fully examine melphalan resistance in patient specimens and define personalized strategies to optimize the use of high-dose melphalan.

Keywords: LC−MS metabolomics; RNAseq; activity-based protein profiling (ABPP); metabolism; multiple myeloma; pentose phosphate pathway; proteometabolomics; purines.

Figure 1:. Experimental Workflow and Qualification of Melphalan Resistant Cell Lines to Rule out Drug Efflux or Drug Metabolism as Resistance Mechanisms.

Proteometabolomics of naïve and derived drug resistant cell lines was used to investigate changes in metabolism in multiple myeloma cells; pharmacological inhibition of selected enzymes and transcriptomics of multiple myeloma tumor cells from newly diagnosed and relapsed patients were used to evaluate the mechanism and assess potential clinical utility. Ion signals for intracellular levels of melphalan as well as its monohydroxylated and dihydroxylated metabolites were summed; ratios of this total ion signal value in drug resistant cells versus naïve cells show that drug efflux pumps do not significantly contribute to melphalan resistance for 8226 and U266 model systems (B and C, respectively). This metric of exposure shows no overall difference in the levels of melphalan and melphalan metabolites over the first 24 hours after treatment. Furthermore, drug resistant cell lines did not detoxify melphalan faster than their respective naïve cell line. At early time points, drug resistant cells appeared to detoxify melphalan slightly more slowly than naïve cells (D and E). See also Supplemental Table S3. * p < 0.05 and ** p < 0.01.

Figure 2:. Steady-state Metabolomics Reveals Metabolites and Pathways Associated with Melphalan Resistance.

Five replicates of naïve and drug resistant cells were analyzed with LC-MS metabolomics. Using log₂ ratio values to compare metabolite levels in naïve and resistant cells, known metabolites from library matching observed with levels >1 standard deviation from the mean log₂ ratio of the known metabolites and with a p-value < 0.05 are visualized in heat maps to compare the largest and most consistent differences in metabolites in the 8226 (A) and U266 (B) model systems. Red signifies metabolites that are observed at higher levels in drug resistant cells and blue indicates lower levels in resistant cells. MetaboAnalyst 4.0 was used to define pathways associated with these metabolites for the 8226 (C) and U266 (D) model systems. Data are presented in Supplemental Table S3. Additional details for the MetaboAnalyst results are shown in Supplemental Figure S8.

Figure 3:. Common Metabolic Changes in Melphalan Resistance in both 8226 and U266 Model Systems Indicate the Importance of the Pentose Phosphate Pathway.

A diagram of the pentose phosphate pathway and guanine salvage has been annotated heat maps for each detected metabolite; if the metabolite was not detected, no data are shown. Font color is used to indicate whether a metabolite is higher in resistant cells (red), lower in resistant cells (blue), unchanged in acquired drug resistance (black), or undetected (gray). Protein ellipses indicate the results from ABPP for 8226/LR5 (left half) and U266/LR6 (right half); both annotated proteins were labeled more in LR5 cells, but not in the LR6 cells, when compared to their naïve counterparts. Asterisks indicate isomers that are indiscernible by LC-MS. Ion signal log₂ values in the bar graph represent PRPS1 (K194_LNVDFALIHK(de)ERK) and HPRT1 (K103_LK(de)SYCNDQSTGDIK) ATP probe uptake in naïve cells 8226 and U266 (N) and resistant LR5 and LR6 (R).

Figure 4:. Levels of Purine and Glutathione Metabolites are Altered in Melphalan Resistance in LR5 Cells.

Proteometabolomics data combining untargeted metabolomics and activity-based protein profiling with ATP probes were mapped on the purine synthesis pathway to illustrate differences between naïve 8226 cells and LR5 drug resistant cells (A). Other than guanine and guanosine, differences between naïve and resistant cells in this model system are not recapitulated in U266/LR6 model system, yet other changes are noted in this pathway (see Supplemental Figure S10 for comparison). Metabolites are shown in rectangles, while proteins are listed as text associated with the arrows denoting relationships between metabolites; both are annotated with the log₂ ratio followed by the p value. |Log₂ ratio| > 0.5 and a p value < 0.05 or a |log₂ ratio| > 1 were considered changed. Red indicates higher values in drug resistant cells, blue shows lower values in drug resistant cells, and white is used when no significant difference was observed. N/A is used for the p value when the number of measurements were insufficient for a t test. Metabolites and proteins in bold font and arrows with heavier line thickness represent potential metabolic reprogramming of purine synthesis and salvage to maintain GMP levels in melphalan resistant cells. To further support the proteometabolomics data, Western blots were used to assess levels of guanine deaminase, GDA (B) and xanthine dehydrogenase/oxidase, XDH/XO, (C) for 8226 and LR5 cells with bar graphs presenting the densitometry data from 3 replicates. Heat maps (D) show relative levels of purine metabolites in 8226 (left) and LR5 (right) cells up to 24 hours after acute melphalan exposure at the LD50 of the naïve cells. Ion signal intensity (relative abundance) is normalized to the average naïve cell pretreatment controls. Red indicates increased levels from the 0-hour time point in naïve cells, white is unchanged, blue is decreased levels, and grey is not detected.

Figure 5:. Multiple Myeloma Cell Lines are Sensitive to Pentose Phosphate Pathway and Tumor Cell Death is Increased by Combination Treatment with Melphalan and 6-Aminonicotinamide.

Cell viability assays for six MM cell lines treated with 6-aminonicotinamide (6-AN) show LD₅₀ values in the low micromolar range (A). Melphalan-resistant LR5 cells are more sensitive to 6-AN than 8226 naïve cells (B), but no difference is observed between LR6 and U266 cells (C). Combination treatment with melphalan and 6-AN in combination does not improve efficacy in 8226 or U266 naïve cells but does in LR5 and LR6 melphalan-resistant cells (D and E, respectively).

Figure 6:. Expression of Key Pentose Phosphate, Guanine Metabolism, and Glutathione Pathway Genes is Higher in Relapsed Myeloma Patients After Melphalan Therapy than in MM cells from Newly Diagnosed Treatment-Naïve Patients.

RNAseq was performed on purified myeloma cells from therapy-naïve patients, N, (n = 177) and specimens taken at relapse following high-dose melphalan therapy, R, (n = 52). Time from therapy to relapse was 2 to 62 months; all maintenance regimens are considered. Relative gene expression is normalized data expressed as z-scores. Solid lines represent the median and dashed lines indicate quartiles. The p values were determined by two-sided Student’s T tests. Not significant is indicated by n.s.