The Polymeric Drug Pamd-Ch17 Induces Mitochondrial Dysfunction and Has Anti-Leukemic Effects

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are deadly blood cancers with survival rates of ~30% in adults. The survival rate is >70% in children, but the harsh chemotherapeutic regimen often leaves patients with significant life-long health complications. Moreover, current chemotherapeutics are inefficient at targeting leukemia stem cells (LSCs) within the bone marrow; this can result in difficult to treat relapse/refractory disease. There is a clear need for improved and less toxic therapeutics. We have developed a new class of polymeric drugs (PAMDs) that have the potential to meet this need. PAMDs are synthesized by Michael addition of AMD3100 and can form nanoparticles. The AMD3100 moiety inhibits CXCR4, leading to mobilization of LSCs to the blood and, potentially, chemosensitization. The polymeric form allows PAMDs to form nanoparticles and encapsulate nucleic acids like siRNAs. Thus, PAMDs have the potential to be modular therapeutics against leukemia. One variant, PAMD-Ch17, is capable of these two functions, but surprisingly, it has additional anti-leukemic effects. Against a panel of human AML and ALL cell lines, PAMD-Ch17 induced a dose-dependent decrease in metabolic activity, with EC50s in the low micromolar range. When tested on mouse primary AML cells, PAMD-Ch17 induced cell death and differentiation, but also significantly reduced colony formation as compared to healthy bone morrow treated with the same concentration of PAMD-Ch17. Consistent with this, a pilot study in vivo showed that PAMD-Ch17 was well-tolerated without toxicity against healthy cells, but markedly reduced leukemic burden. To investigate the mechanism of PAMD-Ch17's anti-leukemic effects, we performed whole transcriptome sequencing (RNA-Seq). Ingenuity Pathway Analysis of differentially expressed genes suggested that PAMD-Ch17 dysregulates pathways related to mitochondrial homeostasis. Confocal microscopy using a fluorescently tagged PAMD-Ch17 showed accumulation of the drug as puncta at the mitochondria, suggesting that PAMD-Ch17 directly targets this organelle. To test this, we performed Seahorse Assays and found that PAMD-Ch17 impairs mitochondrial baseline respiration, ATP-linked respiration, and the proton gradient. To determine how PAMD-Ch17 induces mitochondrial dysfunction, we performed flow cytometry on AML (Molm-13s, Kasumi-1s, ME-1s) and ALL (Jurkats, Molt-4s, SEMs) cell lines. We found that PAMD-Ch17 induced a dose-dependent increase in mitochondrial superoxide, decrease in mitochondrial membrane potential, and increase in cell death. In all the tested cell lines, we observed the increase in superoxide at earlier time points than the changes in mitochondrial membrane potential or cell death, implying that generation of mitochondrial superoxide is the basis of PAMD-Ch17's anti-leukemic effects. Targeting of mitochondrial respiration has not been reported for PAMD-Ch17's parent molecule AMD3100 or for other CXCR4 inhibitors, raising the possibility that PAMD-Ch17's effect on the mitochondria is through a CXCR4-independent mechanism. To test this, we used Crispr/Cas9 to knockout (KO) CXCR4 in both Jurkat and Molm-13 cells. We found that PAMD-Ch17 induces equivalent levels of mitochondrial superoxide and cell death in CXCR4 KO cells, as compared to wildtype. These results indicate that PAMD-Ch17's anti-leukemic effects are CXCR4 independent. Our results suggest that PAMD-Ch17 accumulates in the mitochondria and induces superoxide, leading to mitochondria dysfunction and cell death via a CXCR4 independent mechanism. Leukemia cells are known to have increased reliance on mitochondrial respiration as compared to healthy hematopoietic cells, which may explain PAMD-Ch17's ability to target leukemia cells with minimal effects on healthy cells. Collectively, our findings indicate that PAMDs have the potential to be effective therapeutics for the treatment of leukemia.