Abstract
Abstract 659
The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays a critical role in a variety of tumor cells including hematological malignancies. Class IA PI3Ks are heterodimers that consist of a p85 regulatory and a p110 catalytic subunit. There are several isoforms of both the catalytic (p110α, p110β and p110δ) and regulatory subunits. The p110α isoform of the class IA PI3-Ks was recently genetically validated as a promising target for anticancer therapy. To date, only one compound (imidazo[1,2-a]pyridine, PIK-75) has been described as a very potent and selective inhibitor of this isoform (>100-fold selectivity over p110α and p110δ). In acute myeloid leukemia (AML), aberrant PI3-K activation is detectable in most of cases both in leukemic bulk and in the immature compartment of the leukemic clone. This activation contributes to cell growth, proliferation, survival, and drug-resistance. Furthermore, we have previously shown that the level of Akt phosphorylation on threonine 308, the major target of PI3-K, is correlated with poor outcome in AML patients (Gallay et al, Leukemia 2009, 23(6):1029-38). Therefore, effective targeting of this pathway with pharmacological inhibitors could improve therapeutic outcome in AML. Here, we studied the anti-neoplastic activity of several inhibitors of the PI3-K p110 subunits in AML cell lines and primary patient specimens.
Treatment with PIK-75 led to a decrease of the proliferation in all cell lines at low dose (MTT assay, IC50: 62 nM, 144 nM, 173 nM in KG1, HL60 and KG1a cell lines, respectively). This inhibition of proliferation was due to massive apoptosis of KG1 and KG1a cells in both liquid culture but also after adhesion of leukemic cells on a fibronectin matrix. By contrast, p110ß (TGX221, 10 μM) and p110γ (AS252424, 10 μM) inhibitors only slightly decreased cell proliferation in KG1 and HL60 cells while p110δ inhibitor (IC87114) has no effect up to 10 μM. PIK-75 inhibited the phosphorylation of Akt on Thr308, and downstream effectors (4-EBP1 and RPS6) in these cells. These results strongly suggest a major role of p110α subunit which is highly express in AML cell lines and 19/19 patients samples. Next, we assessed the PIK-75 efficacy in 1 AML cell lines, 9 AML samples and 1 normal bone marrow CD34+ cells using clonogenic assays. PIK-75 inhibited AML-CFU in both KG1 cell line and all patient samples tested with an IC50 of 214 nM and 72 nM, respectively. Interestingly, PIK-75 has no effect on normal CFU-GM colonies, even at high dose (IC50 not reached at 1 μM), a result consistent with the normal haematopoiesis observed in p110αfnKO mice (Gritsman et al, Blood (ASH Annual Meeting Abstracts) 2009 114: Abstract 3620). Since leukemic subpopulation bearing the CD34+CD38-CD123+ phenotype is thought to be more resistant to chemotherapy than the leukemic bulk, we have assessed the apoptosis of 17 AML primary cells treated with increasing doses of cytarabine (Ara-C). We found significant differences of IC50 with 9.5 μM and 43 μM in bulk and CD34+38-123+ subpopulation, respectively. By contrast, PIK-75 demonstrated potent activity in both leukemic compartments of 42 AML samples with IC50 of 589 nM and 638 nM, respectively. Interestingly, the effect of PIK75 was not altered at relapse neither in bulk (IC50: 513 nM vs 492 nM at diagnosis and at relapse) nor in CD34+38-123+ subpopulation (IC50: 567 nM vs 254 nM at diagnosis and at relapse) in 8 AML samples. In NOD/SCID mice engrafted with HL60 cells, PIK-75 delivered at 1 and 10 mg/kg/d for 4 days induced a significant decrease in tumor burden after apoptosis induction detected ex vivo by annexin V staining. Further in vivo studies using NSG mice engrafted with primary AML specimens are ongoing.
These results demonstrate that PIK-75 is the most potent inhibitor of PI3-K in leukemic cells suggesting that the selective inhibition of the p110α subunit could be a critical target in AML. Moreover, PIK-75 targets both leukemic bulk and chemoresistant leukemic subpopulations paving the way for clinical studies assessing the combination of selective p110α inhibitor with conventional chemotherapy.
Disclosures:
No relevant conflicts of interest to declare.
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