Metabolic rewiring is a cancer hallmark. Recently, we reported that in B cell lymphomas, MYC directly regulate three mitochondrial enzymes (IDH2, D2HGDH and L2HGDH) that control alpha-ketoglutarate (αKG) homeostasis (Qiu et al, Cell Chem Biol 2020; Lin et al, Leukemia 2022). Earlier, we showed that in diffuse large B cell lymphomas (DLBCL), αKG can also be deregulated by loss-of-function mutation in D2HGDH (Lin et al, Nat Commun 2015). In parallel, others showed that αKG can suppress colorectal cancer (CRC; Tran et al, Nat Cancer, 2020). Here we examined if αKG has anti-lymphoma activity. We exposed 12 DLBCL cell lines to cell-permeable formulations of αKG (dimethyl-αKG [DMαKG] or octyl-αKG) and detected a dose and time dependent suppression of cell proliferation (mean = 72% vs. vehicle-treated cells, range 47%-99%) and induction of apoptosis (mean = 60% vs. vehicle-treated cells, range 28%-93%). These effects were confirmed in primary B cell tumors tested ex vivo, while normal mature B cells were insensitive to αKG. In CRC, αKG inhibited tumorigenesis by suppressing β-catenin signals downstream of TET activation. Thus, we generated and examined lymphoma models of TET2 KO and constitutively active β-catenin. We confirmed that αKG induces DKK4, a negative regulator of Wnt/β-catenin pathway, in a TET2-dependent manner, but genetic modulation of this pathway had no impact on αKG inhibitory effects in lymphomas, confirming the limited role β-catenin in lymphoma biology. Next, we tested if a feedback loop, which typifies many metabolic pathways, was operational, i.e., if the MYC-promoted αKG generation that we described (Qiu et al, Cell Chem Biol 2020) could be countered by an αKG-mediated suppression of MYC. We found that αKG induced a rapid decrease in MYC protein expression, which was blocked by proteasome inhibitors. Notably, in selected models, an increase in MYC phosphorylation (T58/S62) following exposure to αKG was also detected. To explore these data further, we created genetic models of MYC WT or mutant (T58A/S62A) in P493-6 cells, used tetracycline to suppresses the "endogenous" MYC, and compared the response to αKG in cells exclusively expressing ectopic MYC WT or T58A/S62A mutant (which cannot be phosphorylated and "tagged" for proteasomal degradation). MYC T58A/S62A-expressing cells were significantly more resistant to αKG than MYC-WT models (20% vs. 50% growth suppression, respectively). Concordantly, αKG fully suppressed MYC WT but it did not MYC T58A/S62A mutant. These data suggested that αKG inhibits lymphoma growth at least in part by promoting MYC suppression, possibly via augmented proteasome degradation. However, as the MYC mutant blocked αKG actions only partially, we posited that αKG may suppress other lymphomagenic nodes. To explore this possibility, we first showed that in DLBCL αKG suppressed ATP synthase activity (and ATP levels), as reported earlier in C. elegans (Chin et al, Nature 2014). This fuel stress resulted in broad inhibition of mTORC1, in DLBCL cell lines and primary tumors, in part due to activation of AMPK. Whether the suppression of MYC contributes to mTORC1 inhibition and vice-versa, remains to be elucidated. We next tested the anti-lymphoma activity of αKG in vivo. First, we used an adoptive transfer assay of Eµ-Myc-derived B cell lymphoma. In two independently cohorts (n=30), we examined tumor burden and survival as primary endpoints. The mice were randomized to daily intra-peritoneally (IP) injections of αKG (400mg/day) or vehicle control. Tumor burden was significantly suppressed in the αKG arm (lymph node weights 11mg vs. 21mg, p=0.02) and, in a second cohort, mice in the αKG arm survived significantly longer (median survival = not reached vs. 42 days in control group, p=0.03, HR 0.28, 95% CI 0.07-1.11, logrank). Lastly, in 3 independent cohorts of xenograft models of human DLBCLs, αKG dosed IP (200mg - 400mg, 5 days/week) significantly inhibited subcutaneous tumor growth (p<0.01, n=22). We concluded that αKG via inhibition of at least two oncogenic nodes, MYC and mTORC1, displays anti-lymphoma properties in vitro and in vivo. This discovery has concrete paths for clinical translational as dietary supplementation of αKG was shown to counter inflammation and increased longevity in mice (Shahmirzadi et al, Cell Metab 2020), a result that has spearhead human clinical trials.
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Open Access
Sep 20, 2023
Chuquan Shang + 5
