Integrating Metabolomics and Molecular Pathways to Uncover Therapeutic Vulnerabilities in Richter’s Transformation

Despite advances in targeted therapies, the aggressive transition of chronic lymphocytic leukemia (CLL) to Richter’s transformation (RT) remains an unmet clinical need. While oxidative phosphorylation (OXPHOS) shaped by B-cell receptor signaling is a recognized feature in CLL and RT, metabolism changes driving RT progression are poorly understood. We recently developed an RT murine model by B-cell-restricted knockout of Mga, a negative regulator of MYC, in LSK cells derived from Cd19Cre-Cas9-del(13q)-Sf3b1-K700E mice. Murine RT cells exhibit high MYC and Ki67 expression, upregulated Mga targets Myc, Nme1 (Nucleoside diphosphate kinase), and elevated OXPHOS and lipid accumulation. Targeting MYC and OXPHOS with CDK9i-inhibitor (AZ5576) and OXPHOS complex IIi (TTFA- Triflurothenoylacetone) significantly improved overall survival in murine RT, highlighting the Mga-Myc-Nme1 axis and OXPHOS as viable targets in Mga KO-driven RT (Iyer et al., Sci. Transl. Med, 2024, Iyer et al, ASH, 2023).

To delineate the metabolite changes from CLL to RT, we performed untargeted metabolomics profiling using murine CLL and RT cells. Our results revealed that fatty acid intermediates, glycerol-3P (Gro3P) (FC=11.7) and carnitine (FC=4.3), were significantly (p<0.01) elevated in RT cells. This is coupled with increased expression of the regulatory enzyme Pgp (phosphoglycolate phosphatase) in both murine and human RT samples, suggesting a potential role of MGA deletion in driving aberrant glycerolipid metabolism in RT. Given the heightened lipid accumulation and OXPHOS dependency in RT cells, we hypothesized that linking these metabolic pathways would unveil new vulnerabilities and therapeutic targets for this aggressive disease.

To translate murine findings to human disease and define the molecular regulation for the metabolic changes, we performed untargeted metabolomics in human B cell lines (Nalm6, MEC1) with or without MGA KO. Fatty acid intermediates, Gro3P (FC=3.5), carnitine (FC=22.1), glutathione (g-glu-cys, FC=2.8), and the pentose phosphate pathway (IMP, FC=4.9; Glucuronic acid FC=4.4), were all significantly upregulated in MGA KO Nalm6 cells. Glycerolipid metabolism pathway was highly correlated (r=0.51, p=0.02) in MEC1 and Nalm6 cells with MGA deletion. Moreover, MGA KO cells showed decreased maximal respiration when treated with etomoxir, a fatty-acid oxidation inhibitor, indicating their reliance on lipid substrates. Further analysis using a labeled ¹³C-glucose-based isotope tracing assay revealed that MGA KO Nalm6 cells displayed an increased labeling of TCA cycle substrate citrate (FC=12 vs. 2.6, p < 0.001) and a higher labeled fraction of Gro3P (FC=59.4 vs. 36.3, p < 0.01) compared to control cells, highlighting a strong connection between the TCA cycle and glycerol metabolism upon MGA deletion.

Gro3P is a key metabolite linking glycolysis, glycerolipid synthesis, and OXPHOS via the mitochondrial electron transfer shuttle. To delineate the role of Gro3P in linking the TCA cycle and glycerol metabolism in MGA KO, we examined its impact on lipid synthesis and OXPHOS. Treatment of MGA KO cell lines (MEC1) with Gro3P (500μM) increased lipid droplet formation, elevated the NADH/NAD+ ratio, and enhanced the expression of MYC, NME1, PGP, and mTOR signaling, indicating Gro3P as a critical regulator for lipid metabolism and OXPHOS via the MGA-MYC-NME1-PGP axis. As PGP is a known enzyme converting excessive detrimental Gro3P to less harmful glycerol, we overexpressed PGP in MEC1 cells to examine its role in mediating the crosstalk between OXPHOS and lipid metabolism. Overexpression of PGP increased glycerol levels and lipid accumulation, elevated oxygen consumption, and activated mTOR signaling, confirming PGP as a metabolic gene target. Given the availability of the PGP inhibitor CP1, we examined the therapeutic benefits of combining CP1 with the mitochondrial OXPHOS complex II inhibitor TTFA on murine RT in vivo. The combination improved overall survival (n=8 per group, p < 0.0001) compared to single treatments by reducing mTOR signaling, MYC, and NME1, highlighting the importance of concurrently targeting mitochondrial OXPHOS and glycerolipid metabolism in RT treatment.

Our studies reveal Gro3P as a metabolite driver of OXPHOS in MGA KO RT via the Mga-Myc-Nme1-Pgp axis. Combining glycerolipid metabolism and OXPHOS targeting may offer effective strategies to conquer RT.