Glycogen synthase kinase 3beta 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 beta-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving beta-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/beta-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3beta, axin 1, beta-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3beta 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 GSK3beta have enhanced beta-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3beta 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 GSK3beta in GMP LSC, enabling unphosphorylated beta-catenin to participate in LSC self-renewal. Missplicing of GSK3beta represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target.

Fig. 1.

BC CML progenitors (CD34⁺CD38⁺Lin⁻) transplant leukemia. (A) Mice transplanted with progenitors show signs of leukemia including wasting, piloerection, and lethargy by 6 weeks posttransplantation. (B) Transplantation of progenitors resulted in prominent tumor bioluminescence as demonstrated at 7 weeks posttransplantation. (C) FACS analysis of tumors derived from BC CML progenitors demonstrated 87.4% human engraftment consisting of 29% human myeloid cells (n = 3 experiments). (D Upper) Tumor derived from a mouse transplanted with 1 × 10⁵ progenitor cells. Hematoxylyn-eosin-stained tumor tissue revealed prominent infiltration with human myeloid cells as typified by the human immature granulocyte characteristic of a BC CML granulocytic sarcoma. (Lower) RT-PCR P210 BCR-ABL analysis of hematopoietic tissues including thymus (T), spleen (S), liver (L), bone marrow (B) and tumors (T1-T6) from CD34⁺CD38⁺ transplanted mice (n = 4).

Fig. 2.

Leukemia stem cells are enriched in the BC GMP population. (A) In 8 experiments involving normal bone marrow or cord blood (n = 30 mice) and 12 BC CML experiments (n = 43 mice) equivalent numbers (10³-4 × 10⁵) of HSC, progenitor and blast (Lin⁺) cells per experiment were transplanted. Bioluminescence imaging demonstrated that BC CML GMP had the greatest engraftment potential. (B) In 3 experiments, quantitative bioluminescence engraftment analysis demonstrated that GMP had a higher level of bioluminescence (P = 0.02; asterisk in figure; two-tailed Student's t test) than HSC (P = 0.06) or Lin⁺ (P = 0.35). (C Left) FACS analysis of tumors (n = 9) from mice transplanted with 2° BC CML progenitors demonstrated a preponderance of GMP (66.4%; P = 8.6 × 10⁻⁹; two-tailed Student's t test) while common myeloid progenitors (CMP) (16.3%) and megakaryocyte-erythroid progenitors (MEP) (2.2%) represented a minority of cells. (Middle) Tertiary (3°) BC CML GMP transplantation of 1 × 10³, 5 × 10³, 1 × 10⁴ and 5 × 10⁴ resulted in engraftment of a CD45RA positive progenitor population in transplanted mice (n = 3 experiments). (Right) Graph of 3° BC CML GMP titration experiments. (D) Bioluminescence imaging was performed 9 weeks posttransplantation and demonstrated that both 2° normal HSC and 2° BC CML GMP (n = 6 mice) had long-term engraftment capacity but 1° normal GMP did not. Primary normal cord blood CD34⁺ cells served as a positive control for engraftment. (E) RT-PCR analysis of P210 BCR-ABL expression in livers from transplanted mice revealed that 2° myeloid BC GMP harbored P210 BCR-ABL transcripts. There were no detectable BCR-ABL transcripts in mice that were untransplanted or those that were transplanted with normal GMP, normal HSC, 2° normal HSC, or lymphoid BC GMP from P190 BCR-ABL-expressing marrow.

Fig. 3.

Aberrant GSK3β expression by BC CML progenitors. (A) FACS plot demonstrating characteristic expansion of the GMP compartment in BC CML compared with normal blood and marrow samples. FACS analysis performed on normal (n = 9) and CML CP (n = 5), CML AP (n = 6), and BC CML (n = 6) blood and bone marrow samples revealed that, while the proportion of HSC did not expand with progression to BC, there was a significant increase in GMP compared with normal controls (P = 1.93 × 106; two-tailed Student's t test). (B) HSC and progenitors were FACS sorted from normal or BC CML CD34⁺ (n = 3) blood samples and GSK3β transcript levels measured by quantitative RT-PCR. There was a significant difference (P < 0.05; two-tailed Student's t test) in GSK3β transcript levels between normal (mean 0.98 ± S.E.M. 0.05) and BC CML progenitors (0.36 ± S.E. 0.04). (C) FACS histograms of CML CP (n = 3), AP (n = 2), and BC (n = 3) progenitors revealed a decrease in GSK3β protein expression with progression to BC. (D) FACS analysis performed on normal blood (n = 5), and BC CML (n = 5) revealed that there was not a significant difference (P = 0.58; two-tailed Student's t test) in GSK3α protein expression as measured by mean fluorescence intensity ± SEM in BC CML samples compared with normal blood. Mean fluorescence intensity of isotype control (Rabbit IgG) was subtracted from all of the samples. (E Left) FACS analysis performed on normal blood (n = 6), and BC CML (n = 5) revealed that there was a significant difference (P = 0.016; two-tailed Student's t test) in activated β-catenin levels as measured by mean fluorescence intensity ± SEM in BC CML compared with normal blood. Isotype control (Mouse IgG₁) was subtracted from all of the samples. (Right) Confocal fluorescence microscopic analysis revealed that normal GMP had little activated nuclear β-catenin whereas BC CML GMP expressing misspliced GSK3β had high levels of nuclear β-catenin (green: CD45 membrane marker, blue: Hoechst nuclear stain, red: activated β-catenin). (F) In 6 experiments, BC GMP from CML samples (n = 2) or tumor (n = 1) derived from BC GMP transplanted mice and normal cord blood GMP (n = 3) were transduced with a lentiviral LEF/TCF GFP reporter for activated β-catenin. BC GMP samples had significantly higher GFP expression (P = 0.037, two-tailed Student's t test) than normal cord blood GMP (n = 3) treated in the same manner. Results are expressed as percentage of maximum fluorescence intensity. (G Left) BC CML HSC in 5 of 8 patient samples subjected to cDNA sequencing analysis had demonstrable misspliced GSK3β transcripts. Nucleotide sequence data represents 2 species of GSK3β transcript in HSC: misspliced GSK3β and FL-GSK3. (Middle) BC CML progenitors in 5 of 8 samples had prominent misspliced GSK3β transcripts in the ORF of the cDNA. (Right) BC CML lineage-positive (blast) cells showing a deletion of GSK3β exon 9 in the ORF of the cDNA that was also detectable in normal samples.

Fig. 4.

Enhanced engraftment of misspliced GSK3β-expressing CML progenitors. (A) P210 BCR-ABL RT-PCR transcript levels in hematopoietic tissues from mice transplanted with human CD45⁺ cells sorted from BC CML progenitor transplanted mice (10⁵ cells; n = 7 mice). Tissues include thymus (T), spleen (S), liver (L), bone marrow (B) and tumors (T1-T5) (B) FACS analysis of 2° myeloid progenitor engrafted mice transplanted with human CD45⁺ cells sorted from bone marrow, liver, and spleen of mice that was originally transplanted with BC CML progenitor cells (n = 3 experiments). (C) Western blot of K562 cells (size control, left lane) transduced with GSK3β full length (FL), GSK3β exon 9 deleted (Exon 9), or GSK3β exon 8 and 9 deleted (m) and of 34⁺CD38⁺ CML BC derived tumor (right lane), shows that mGSK3β protein is expressed in the tumor cells. (D) Representative FACS plots of long-term (week 11–12) human CD45 and CD14/33 bone marrow (BM) engraftment in no transplant control (Left), BC progenitors transduced with FL-GSK3β (Middle) and misspliced GSK3β (n = 3) transduced BC CML progenitor transplanted mice (Right). (E) FACS analysis revealed a decreased percentage of long-term human CD45 cells in the PI negative fraction ± S.E.M. in hematopoietic organs including bone marrow (B; P = 0.019), liver (L; P = 0.025), spleen (S; P = 0.0109), and thymus (T; P = 0.10) of mice transplanted with FL-GSK3β (n = 3) compared with m-GSK3β transduced (n = 3) BC CML progenitors. Reduced human CD14 and CD33 engraftment was also noted in bone marrow (P = 0.03), liver (P = 0.08), spleen (P = 0.024), and thymus (P = 0.22) while there was no appreciable engraftment of human CD3 or CD19 cells in BC CML progenitor transplanted mice.