Glycogen synthase kinase 3β missplicing contributes to leukemia stem cell generation

Abstract

Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase (CP) in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis (BC), characterized by aberrant activation of ␤-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving ␤-catenin activation. Here we show that BC CML myeloid progenitors, in particular GMP, serially transplant leukemia in immunocompromised mice and thus are enriched for leukemia stem cells (LSC). Notably, cDNA sequencing of Wnt/␤-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3␤, axin 1, ␤-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3␤ kinase domain in the GMP of BC samples that was not detectable by sequencing in blasts or normal progenitors. Moreover, BC CML progenitors with misspliced GSK3␤ have enhanced ␤-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3␤ reduces both in vitro replating and leukemic engraftment. We propose that CP CML is initiated by BCR-ABL expression in an HSC clone but that progression to BC may include missplicing of GSK3␤ in GMP LSC, enabling unphosphorylated ␤-catenin to participate in LSC self-renewal. Missplicing of GSK3␤ represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target.blast crisis chronic myeloid leukemia ͉ wnt pathway ͉ xenograft ͉ self-renewal ͉ cancer stem cells

How One Thing Led to Another

“…It should be pointed out that the leukemogenic splice form of GSK3β could hardly be seen in arrays of unpurified CML blast crisis bone marrow, insofar as the LSCs were only ∼5% of all leukemic marrow cells (200). This provides a very important caution to those who would do epigenetic analyses such as microarrays, RNAseq, chromatin immunoprecipitation, and DNA CpG methylation, to name a few.…”

“…We showed that in 4 of 7 patients the activation of the β-catenin pathway occurred because of GMP stage-specific missplicing of GSK3β (200), the cytoplasmic enzyme held in a scaffold complex (with β-catenin, APC, and axin), which phosphorylates β-catenin to tag it for ubiquitination and cytoplasmic proteasomal destruction. The GMP-LSC splice form was uniformly lacking exon 8, encoding the kinase domain, and this was the only splice form in the GMP LSC (200). Both HSCs and Lin + blast cell progeny of the LSCs expressed the splice forms containing exon 8 (200).…”

“…Both HSCs and Lin + blast cell progeny of the LSCs expressed the splice forms containing exon 8 (200). We could block proliferation of these LSCs by transfection of either axin or the cDNA of full-length GSK3β (163,200).…”

Insights into the stem cells of chronic myeloid leukemia

“…[39][40][41] The variable, but generally long, phase that precedes the diagnosis of chronic phase CML also suggests a high variability in the precise stage of the disease when the diagnosis will be made. This variability is unquestionably further enhanced by the fact that more and more CML patients are being diagnosed by chance (before they have become symptomatic) as part of a routine check-up that reveals an elevated white blood cell count.…”

“…40 It is interesting that these findings add to the accumulating experimental evidence that certain mutations are sufficient to reactivate or confer self-renewal activity upon cells in which such programs had previously been thought to be normally extinguished. 38,39,41,[46][47][48] The anatomy of primitive elements in chronic phase CML clones…”