Abstract
The introduction of BCR-ABL tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML). However, although the majority of patients with chronic phase (CP)-CML obtain durable complete cytogenetic and major molecular responses, there is low level disease persistence postulated to be due to a population of TKI-insensitive leukemia stem cells (LSC). The aims of this study were (1) to fully characterize differences in gene expression between normal hematopoietic stem cells (HSC) and CP-CML LSC and (2) identify potential novel therapeutic targets specific to CML LSC.
Lin-CD34+CD38- CD45RA-CD90+ normal HSC (n=3) and CP-CML LSC (n=6 patients at diagnosis), populations were isolated using a FACSAria and applied to Affymetrix HuGene 1.0ST arrays. The raw data (.CEL files) was imported into Partek Genomics Suite and Ingenuity Pathway Analysis software and principal component analysis and gene ontology ANOVA performed. A total of 1217 genes were significantly deregulated between normal HSC and CP-CML LSC. The most significantly deregulated genes and pathways were involved with the molecular and cellular functions of cell cycle, cell assembly and organisation, cellular movement, cell death and DNA replication, recombination and repair. These results suggested that CML LSC were less quiescent than normal HSC. Importantly, complimentary functional studies indicated that CML LSC have significantly increased proliferation (14 fold expansion; P<0.001) compared to normal HSC (no expansion) after 5 days in vitro culture. In addition, equivalent numbers of CML LSC produce ~4-fold more colonies in colony forming cell (CFC) assays than normal HSC (329±56 versus 86±17 per 2,000 cells, respectively; P<0.05). Fluorescence in situ hybridisation (FISH) demonstrated that >90% of lin- CD34+ CD38- CD45RA- CD90+ CML LSC from all patient samples were BCR-ABL positive (+).
In addition to these deregulated intracellular pathways, we sought to assess if there were differences in expression of cell surface molecules that may be amenable to therapeutic manipulation. Of particular interest, our microarray studies demonstrated that CD93 was highly upregulated in CP-CML LSC (6 fold, p = 2.5x10-6). Increased CD93 expression was validated by Fluidigm digital PCR (6 fold increase, p = 0.02; n=6). Furthermore, using flow cytometry, we demonstrated significant upregulation of CD93 protein expression on lin-CD34+ CD38- CD45RA- CD90+ CML LSC from peripheral blood and bone marrow of CP-CML patients (n= 17; mean = 63.8% CD93+) compared to normal HSC from healthy peripheral blood stem cell donors (n=7; mean = 0.8% CD93+) and bone marrow donors (n=4; mean = 0.2% CD93+; p < 0.0001). FISH confirmed that 100% of lin-CD34+ CD38- CD90+ CD93+ CML cells were BCR-ABL+ in all samples assessed.
CD93 (also known as C1qRp) is a C-type lectin-like domain (CTLD)-containing glycoprotein which regulates phagocytosis, with roles in cell adhesion and leukocyte migration. It is normally expressed on endothelial cells, hematopoietic precursors and mature cells including neutrophils, monocytes and platelets. Previous studies have shown CD93 to be upregulated in a proportion of AML patients (Saito et al, Sci Transl Med, 2010. 2(17): p. 17ra9).
Short term (24h) in vitro exposure of lin-CD34+CD38- CD45RA- CD90+ CML LSC to TKIs (Imatinib or Dasatinib; n=3) reduced, but did not fully eliminate CD93 expression (Imatinib, 48.5% to 22.9%; Dasatinib, 47.7% to 9.2%). Importantly, following long-term TKI treatment of patients, lin-CD34+CD38-CD45RA-CD90+ cells from CP-CML patient bone marrow samples (n=2) taken in major molecular response demonstrated a small, but persistent population of CD93+ LSC which were BCR-ABL+ by FISH. Furthermore, in xenograft transplantation experiments (n = 5), after 16 weeks, CD34+CD93+ CML LSC engrafted lethally irradiated NOD/SCID/IL-2Rg-/- (NSG) mice with BCR-ABL+ cells, whereas CD34+CD93- cells from the same patient samples failed to engraft to significant levels (3.5-30 fold increase in engraftment with CD34+CD93- cells; p < 0.03). FISH confirmed that engrafted human cells were BCR-ABL+.
Taken together, our results identify CD93 as a potential novel biomarker of CML LSC, which may also be helpful in assessing minimal residual disease at the LSC level. Further studies are ongoing to assess the therapeutic potential of inhibiting CD93 in CML LSC.
Disclosures
Copland: Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Anlotinib Potentially Eliminates Leukemia Stem Cells and Modulates the Chemosensitivity via Inhibiting JAK2-STAT3/5 Signaling
