Integrin-Linked Kinase a Novel Therapeutic Target for Acute and Chronic Myeloid Leukemia

Introduction:

Current treatment options as well as clinical efficacy are limited for acute myeloid leukemia (AML), Ph+ acute lymphoblastic leukemia (Ph+ ALL), and chronic myelogenous leukemia (CML). In response to the pressing need for more efficacious treatment approaches and strategies to override drug resistance in advanced stage AML, Ph+ ALL, and CML, we investigated the effects of inhibition of integrin-linked kinase (ILK) as a potentially novel and effective approach to treatment of these challenging malignancies. ILK is an intracellular adaptor and kinase that links the integrins, cell-adhesion receptors, and growth factors to a range of signaling pathways. It has been shown that inhibition of ILK expression and activity is anti-tumorigenic, which makes ILK an attractive target for cancer therapeutics. Compound 22 (Cpd22, N-Methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide) is a cell-permeable, tri-substituted pyrazol compound that acts as a potent and targeted ILK inhibitor. In the present study, we investigated ILK as a putative novel target for treatment of AML, Ph+ ALL, and CML, and Cpd22 as a potential novel anti-leukemia agent.

Methods:

Compound 22 (Cpd22) was purchased from EMD Millipore. The expression of ILK in AML (K052, NOMO-1, THP, MOLM14), CML (K562, Ku812F, LAMA84S and LAMA84R), and Ph+ ALL (SUP-B15) cell lines, primary patient cells, and human Bone Marrow Mononuclear Cells (BM MNCs) was analyzed by flow cytometry. AML, Ph+ ALL, CML cell lines and peripheral blood mononuclear cells (PBMCs) were cultured with ILK inhibitor, Cpd22 (0-1000 nM) for 3 days. Toxicity of Cdp22 (0-500 nM) toward BM MNCs and primary patient cells (CML, ALL, and AML) was also assessed. Cell lines and/or primary patient cells were analyzed for cell proliferation by MTT assay; cell cycle by DNA staining with PI and analyzed by flow cytometry; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with proliferation, cell cycle, and apoptosis was analyzed by western blotting. In addition, ILK knockdown of AML and CML cell lines was evaluated.

Results:

Normal BM MNCs showed ILK expression, but expression was lower than in cell lines and primary samples from AML, CML, and ALL. Cpd22 inhibited the proliferation of AML, ALL, and CML cell lines, while none of normal PBMC and normal BM MNCs controls were affected by the same concentration range after three days of treatment with Cpd22 as a single agent. ILK expression in primary AML patient cells correlated with efficacy of Cpd22: Specifically, the higher the ILK expression, the more sensitivity to Cpd22. ILK expression in primary ALL patient cells also correlated with the efficacy of Cpd22: Specifically, the higher the ILK expression, the more sensitivity to Cpd22. We validated the target specificity of Cpd22 in CML cells by immuno-blotting and investigating inhibitor effects on signaling molecules downstream of ILK. Cpd22 potently suppressed the phosphorylation levels of Ser-473-Akt (pAKT) and another ILK substrate, pGSK-3.

We observed cell cycle arrest in Cpd22-treated cells, specifically accumulation of apoptotic cells in subG1 and Annexin/PI staining showed a 3-fold increase in the fraction of apoptotic cells staining positive for Annexin and PI in Cpd22-treated cells. Immuno-blotting confirmed cell cycle arrest by decreased pRb and increased cell cycle inhibitor p27; and drug induction of apoptosis through the caspase intrinsic pathway by demonstrating increased cleavage of caspase-3 and caspase-9.

The importance of ILK for the growth of leukemia cells was demonstrated by knockdown of ILK in AML and CML cell lines, which led to decreases in cell proliferation. These results suggest a partial dependency of acute and chronic myeloid leukemia cells on ILK for growth, and are consistent with results obtained with the ILK inhibitor, Cpd22.

Conclusions:

In conclusion, our results suggest that the ILK inhibition may be an effective treatment for AML, Ph+ ALL, and CML as a single therapy, with ILK expression levels positively correlating with the efficacy of ILK inhibition. The identification of ILK as a novel target for leukemia therapy warrants further investigation as a therapeutic approach that could be of potential clinical benefit in both acute and chronic myeloid leukemias.