Richter's syndrome (RS) is an aggressive transition of chronic lymphocytic leukemia (CLL) to lymphoma; however, molecular mechanisms underlying CLL-to-RS transformation are poorly understood. MYC network alterations are observed in 70% of RS cases, and MGA (Max-gene-associated), a functional MYC suppressor, undergoes loss-of-function mutations in ~7-20% of RS cases. MYC overexpression induces oxidative stress via reactive oxygen species (ROS) in several B-cell lymphomas. Using CRISPR-Cas9, we recently developed a B-cell restricted CLL to RS murine model by engineering loss-of-function Mga mutations in early progenitors (LSK) in the presence of commonly occurring CLL mutations 13q deletion (Mdr) and Sf3b1K700E. We found Myc and Nme1 (Nucleoside diphosphate kinase), both common targets of Mga, were up-regulated, altering the mitochondrial structure, and increasing oxidative phosphorylation (OXPHOS), leading to mitochondrial dysregulation. Based on these observations, we sought to understand the molecular basis of mitochondrial dysregulation and determine if targeting the Mga-Myc-Nme1 axis is beneficial for RS in vivo. Dysregulated mitochondria produce reactive oxygen species (ROS), byproducts of OXPHOS that are reported to trigger oncogenic signaling. To determine the molecular basis of mitochondrial dysregulation, we used our previously established Mga KO (knockout) driven CLL to RS murine model, which develops RS upon CLL cells transplantation into either immunocompetent or immunocompromised mice with high Ki67, MYC, and CD5 loss along with aberrant changes in mitochondria. Firstly, we measured cellular and mitochondrial ROS in splenic B cells derived from CLL, RS, and healthy control mice (n=3 each). CLL and RS B cells had higher cellular ROS and mitochondrial ROS than normal B cells. RS cells showed an elevated cellular ROS compared to CLL cells (p< 0.001). Accordingly, RS mitochondria exhibited high utilization of TCA cycle substrates such as succinate and malate (Seahorse substrate oxidation test). The substrates for complex II of the mitochondrial electron transport chain were also highly utilized. To unravel the molecular mechanism underlying MGA KO-induced metabolic changes, we generated MGA KO human B cell lines [MEC1, HG3, Nalm6E (harboring SF3B1K700E)] by CRISPR-Cas9. MGA KO increased cellular ROS in all cell lines and upregulated NME1, ETC (electron transport chain) complex II protein levels, suggesting that NME1, and ETC complex II may regulate ROS. Moreover, MGA KO induced pERK1/2 (ERK) and p4E-BP1 (mTOR) activation in an NME1-dependent fashion, which was reversed by antioxidant treatment (N-acetyl cysteine), highlighting MGA KO-induced ROS as a key driver of oncogenic activation. Given that ROS activates oncogenic signaling in MGA KO B cells, we exploited mitochondrial dysregulation as a potential therapeutic approach to treating RS. Firstly, we tested the inhibitor of complex II (TTFA: Thenoyltrifluoroacetone) and CDK9 (AZ5576) in MGA KO Nalm6E cells. TTFA and CDK9 inhibitors significantly decreased the activation of pERK1/2 and p4EBP1(mTOR) with a concomitant decrease in MYC and NME1 at the protein level. We next explored these inhibitors for treating murine RS in vivo. We established murine RS cells by engrafting RS cells into NSG mice and then treated RS mice by orally given vehicle (n = 13); AZ5576 (60mg/kg) (n = 13); TTFA (25mg/kg) (n = 4); and AZ + TTFA (60 and 25mg/kg) (n = 5). AZ5576 and TTFA single treatment significantly prolonged the survival of RS mice (Log-rank test, p < 0.0001) with a reduction in total spleen weight (p=0.0007). AZ+TTFA combination had synergy in increasing the survival of RS mice by 10 days (p=0.0003) compared to a single treatment. AZ+TTFA treated murine RS cells showed reduced MYC, NME1, and p4E-BP1 protein expression, confirming the effectiveness of targeting of Mga-Myc-Nme1 axis. In summary, our murine model reveals that MGA/MYC/NME1 axis drives RS through increased mitochondrial OXPHOS and ROS accumulation. Moreover, targeting this axis provides therapeutic benefits for RS, highlighting this pathway as a novel potential target for RS treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal
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