MGA loss promotes mitochondrial structure changes and redox adaptation in richter's transformation

Abstract Richter's transformation, a highly aggressive and therapy-refractory progression of chronic lymphocytic leukemia (CLL), is characterized by MYC activation and elevated OXPHOS; however, how mitochondrial remodeling contributes to aggressiveness remains unclear. MGA (Max gene-associated), a transcriptional repressor of MYC, is recurrently inactivated by loss-of-function mutations in ~30% of RT cases, leading to MYC overexpression and enhanced metabolic stress. Given MYC's central role in regulating OXPHOS, we hypothesize that MGA loss promotes mitochondrial remodeling to support the metabolic demands and aggressiveness of RT. To dissect the role of MGA in driving RT, we recently developed a B-cell-restricted RT murine model by in vitro CRISPR-Cas9 editing of LSK progenitors from Cd19-Cre-Cas9 del(13q)-Sf3b1 (Sf3b1-K700E) mice using sgRNAs against Mga. This RT murine model recapitulated human RT features, including large cell size, high MYC and Ki67 expression, high cellular reactive oxygen species (ROS), and increased NME1 (MYC-MGA target) and electron transport complex II protein levels compared to CLL and normal B cells. Transmission electron microscopy (TEM) analysis revealed increased mitochondrial cristae width, area, and abnormal shapes in RT cells. Transcriptomic analysis of murine CLL-RT B cells revealed significant (p < 0.01) upregulation (log2 FC (fold change) > 1.5) of oxidative stress response genes (PRDX1, 2, 4, Peroxiredoxin family of antioxidant genes) and MICOS (mitochondrial contact site and cristae organizing system) complex genes in RT compared to CLL, suggesting that mitochondrial and redox adaptations are central to RT biology. Given these observations, we focused on the mitochondrial contact site and cristae organizing system (MICOS) complex, which maintains the inner membrane architecture and integrity of cristae junctions. Disruption of MICOS alters cristae morphology, impairs mitochondrial compartmentalization, and compromises OXPHOS efficiency. Immunoblot analysis revealed significant upregulation of multiple MICOS complex components-MIC60, MIC25 (CHCHD6), MIC19, and MIC27 (APOOL), in murine RT samples compared to CLL and normal B cells. APOOL was one of the significantly upregulated MICOS proteins in murine RT and human RT-PDX samples (n = 3) and MGA KO HG3 cell lines. This coordinated increase in cristae-organizing proteins suggests that RT cells may undergo active mitochondrial remodeling to sustain elevated OXPHOS under metabolic stress. Given the high expression of APOOL in RT cells, we generated APOOL KO human B cell lines (NALM6, HG3) via CRISPR-Cas9 to assess the connection between mitochondrial remodeling and OXPHOS. APOOL KO decreased OXPHOS, reduced cell growth rates, and total NADH in NALM6 and HG3 cell lines and was associated with increased expression of MIC25 and MIC19, decreased phosphorylation of 4E-BP1 (mTOR signaling), and reduced mitochondrial membrane potential. TEM analysis revealed disrupted mitochondrial ultrastructure, characterized by fragmented cristae, reduced cristae density, and altered inner membrane organization, consistent with impaired MICOS function and mitochondrial instability. To further elucidate the response to oxidative stress, we profiled the peroxiredoxin (PRDX) family members, which detoxify ROS and protect mitochondrial function. First, PRDX1 and PRDX2 were upregulated in human DLBCL tumors compared to normal genotype-tissue expression samples in the TCGA data, as determined by gene-expression profiling interactive analysis. Furthermore, PRDX1 and PRDX2 were upregulated in murine RT and human RT PDX samples (n = 3) compared to CLL (n = 3) and normal B cells (n = 4) by immunoblot analysis. As MYC can upregulate PRDX2 expression by binding to the promoter but not PRDX1, we focused on functional studies with PRDX2. PRDX2 KO in human B cell lines (Nalm6 and HG3 cells expressing Cas9) led to reduced APOOL protein levels, cell growth, decreased glucose uptake, suppressed mTOR signaling (p4EBP1), and impaired oxygen consumption rate, highlighting PRDX2 as a critical regulator of redox balance and metabolic fitness in RT cells. Our findings identify a mitochondrial adaptation program in RT, characterized by the upregulation of the MICOS complex and PRDX2, which supports survival under metabolic stress. Targeting cristae structure and redox balance may offer new therapeutic avenues for MYC-driven, treatment-refractory RT.