Abstract
Growing evidence indicates that interactions of cancer cells with their microenvironment in vivo can influence disease progression and therapy resistance, including chronic myeloid leukemia (CML). Focal adhesions that modulate cell attachments, migration, proliferation and intracellular signaling pathways are considered critical mediators of some of these interactions. However, the potential role of focal adhesion components in mediating survival and therapeutic responses of leukemic stem cells is largely unknown.
Transcriptional profiling of CD34+ cells from 6 CML patients and 3 healthy donors revealed that the expression of Integrin-linked kinase (ILK), PINCH1 and β-Parvin, major constituents of focal adhesions, is significantly increased in CD34+ CML cells, in particular in cells from drug-nonresponders (p<0.05). Quantitative real-time PCR confirmed these observations in CD34+ cells obtained from additional 30 CML patients and 14 normal healthy adults (p<0.05). Furthermore, we found that the primitive leukemic stem-cell enriched Lin-CD34+CD38- portion from CML patients expressed the highest levels of ILK, PINCH1, and β-Parvin transcripts compared to the more prevalent Lin-CD34+CD38+ progenitor population or mature CD34-cells in the same samples (n=6, p<0.05). In addition, ILK protein expression was increased in primitive CML cells compared to normal donors, in particular when CML cells were co-cultured with BM niche cells.
Stable knockdown (KD) of 3 different targeting sequences of ILK in CD34+ CML cells resulted in decreased cell viability (30-80%, p<0.05) and proliferation (2-12-fold) associated with a significantly enhanced frequency of apoptotic cells compared to control-transduced cells (60-80% vs. 30%, p<0.05). Interestingly, these effects of ILK KD were not rescued by co-cultures with BM niche cells in vitro. Cell cycle analysis indicated a reduction in the proportion of surviving cells in S-phase upon ILK suppression. In addition, Western blotting showed that effective suppression of ILK led also to a decrease in β-Parvin and PINCH1 protein expression but not their transcript levels, suggesting that the ILK-PINCH-PARVIN complex is not stable under these conditions and may not be able to mediate critical interactions between primitive CML cells and BM niche components. In agreement, short- and long-term assays of stem/progenitor activity in the presence of BM niche cells demonstrated a significant reduction of colonies upon ILK suppression that was almost entirely abolished with simultaneous ABL1 tyrosine kinase inhibitor (TKI) treatment (p<0.05). Moreover, in vivo studies with 2 different mouse strains (NRG and the humanized NRG-3GS model) emphasized that primitive ILK KD CML cells showed greatly reduced in vivo regenerative activity as compared to control-transduced cells (<2% vs. 13% human cells in the BM of NRG mice, and 3% vs. 18% in NRG-3GS mice 25 weeks post-transplantation).
To investigate whether ILK can be targeted pharmacologically, we utilized QLT0267, a validated and selective ILK kinase inhibitor. Similarly to ILK suppression, inhibition of the ILK kinase resulted in a modest decrease of cell viability, reduced short-and long-term stem/progenitor activity, and increased apoptosis of bulk CD34+ as well as more primitive Lin-CD34+CD38- CML cells from drug-nonresponder patients with strong synergistic effects upon simultaneous ABL1 kinase inhibition in vitro. In addition, oral gavage of QLT0267 combined with dasatinib significantly enhanced survival of leukemic mice and eradicated infiltrated leukemic cells in multiple hematopoietic tissues in an aggressive NSG mouse model of BCR-ABL+human leukemia. Most interestingly, dual inhibition of ILK and BCR-ABL1 decreased the proportion of quiescent leukemic stem cells compared to single agent treatments. RNA sequencing of these cells indicated a deregulation of MYC and novel signaling targets, with differences between dividing and non-dividing cell subpopulations.
In summary, genetic and pharmacological inhibition of ILK significantly impaired survival, proliferation and quiescence of drug-nonresponder CML stem cells and sensitized them to TKIs both in vitro and in vivo. These findings suggest that ILK plays a critical role in regulating CML stem cell activity and that targeting ILK and BCR-ABL1 simultaneously may offer an improved novel therapeutic strategy.
Disclosures
No relevant conflicts of interest to declare.
Cell motion as a stochastic process controlled by focal contacts dynamics