Depletion of CD59 Inhibits the Proliferation and Survival of Acute Myeloid Leukemia Cells By Altering Lipid Raft Composition and Suppression of Raf/MEK/ERK Signaling

Treatment outcomes for acute myeloid leukemia (AML) patients vary greatly based on patient characteristics, such as age and the genetic makeup of leukemic cells. In particular, older patients (>60 years), patients with complex cytogenetics, and patients with TP53 mutations are more resistant to frontline treatment and have dismal overall survival. The lack of effective treatment options for this high-risk patient population presents the need to identify novel therapeutic targets. We previously reported that the membrane regulatory protein, CD59, is a marker for poor prognosis in AML, with increased CD59 expression associated with refractory disease, TP53 mutational status, and shorter overall survival. CD59 is a GPI-anchored protein which mainly inhibits the formation of the membrane attack complex of the complement system. Using doxycycline-inducible short-hairpin RNAs against CD59 (shCD59) and a non-targeting control (shNT), we showed that CD59 knockdown leads to G0/G1 cell cycle arrest and increased cell death in AML cell lines. Importantly, we confirmed a complement-independent role of CD59 as the effect of CD59 knockdown is not rescued by heat-inactivation of fetal bovine serum, a procedure which inactivates the complement proteins in cell culture medium. Here, we build upon our findings by testing for in vivo efficacy. We transplanted NOD/SCID/IL2Ry-null (NSG) mice with AML cells which were transduced with doxycycline-inducible shCD59 or shNT-encoding lentiviral vectors. Doxycycline-treated mice transplanted with shCD59-expressing cells had a 1000-fold lower leukemic burden and significantly longer overall survival in comparison to doxycycline-treated mice transplanted with shNT-expressing cells or vehicle-treated mice transplanted with shCD59 or shNT-expressing cells. To investigate the mechanism through which CD59 expression promotes AML proliferation, we performed RNA-sequencing and mass spectrometry-based phospho-proteomic profiling of shCD59 and shNT-expressing AML cells. Differential gene expression and pathway enrichment analyses revealed a downregulation of genes involved in cell cycle and DNA replication upon CD59 knockdown. This indicated that CD59 influences the activation of pathways that control cell cycling. As such, we employed a kinase activity prediction algorithm which predicted a decrease in extracellular-signal regulated kinase 1 (ERK1) activity. As effector proteins of the Ras/Raf/MEK signaling pathway, ERK1/2 are well-documented drivers of G1-S phase transition in AML. We therefore probed for active MEK1/2 and ERK1/2 levels via western blotting and confirmed a decrease in p-MEK1/2 (S217/221) and p-ERK1/2 (T202/Y204) expression following CD59 knockdown, whereas total MEK1/2 and ERK1/2 levels were unchanged. This finding indicated that CD59 signals upstream of MEK1/2 to influence ERK1/2 activation. As CD59 is reported to cluster within lipid rafts, which are important sites for signal transduction, we predicted that CD59 downregulation alters lipid raft signaling. First, we probed for the abundance of the lipid raft marker, GM1, via immunofluorescence microscopy and observed an increase in GM1 staining intensity in shCD59-expressing cells versus shNT-expressing cells. As various proteins compete for lipid raft localization, we reasoned that CD59 depletion, and subsequent increase in GM1 abundance, leads to a change in the protein composition of lipid rafts. Therefore, we isolated the lipid raft and cytosolic fractions from shCD59 and shNT-expressing cells and probed for Ras, c-Raf, and MEK1/2 expression. We observed increased Ras and c-Raf expression in the lipid raft compartment following CD59 knockdown, whereas there was no change in MEK1/2 expression. Furthermore, there was no change in the level of active Ras (GTP-bound Ras) between shCD59 and shNT-expressing cells. Thus, these findings indicate that CD59 depletion increases Ras and c-Raf localization to the lipid raft fraction which prevents MEK1/2 and ERK1/2 activation. In summary, we demonstrate that reducing CD59 expression prevents the proliferation of AML cells in a complement-independent manner. We propose a model in which CD59 depletion alters the composition of lipid rafts, thereby supressing ERK signaling which leads to cell cycle arrest. Our findings provide the rationale for exploring CD59 as a therapeutic target against this deadly disease.

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