Results of the present study provide the first evidence that human AML cells express exclusively isoform A, the proliferative form of the IR. Additional experiments demonstrate that the dual IGF1R/IR inhibitor BMS-536924 decreases autophosphorylation of its target receptors, diminishes signaling through the PI3K/Akt and MAP kinase pathways, and inhibits proliferation of AML cell lines and colony formation in clinical AML samples. Collectively, these results identify the IGF1R/IR axis as a possible contributor to AML cell survival and demonstrate for the potential utility of targeting both receptors in AML.
In earlier work, Tazzari et al
. reported that IGF1 expression and IGF1R phosphorylation were increased in blast cells at the time of relapse in 4 AML patients (29
). Doepfner et al.
subsequently demonstrated expression of IR in addition to IGF1R in AML blasts (28
). The present study extended these observations by showing that both IR and IGF1R are expressed at the protein and RNA level in >80% of AML samples (). When primers that amplify across exon 11 of the IR (6
) were utilized, no exon 11 sequence was detected in any of the AML cell lines () or clinical AML samples (). In view of the ability of IR-A to bind IGF1 and IGF2 directly (7
) as well as modify the ligand specificity of IGF1R (6
), our demonstration that isoform A is the predominant IR expressed in AML has potential biological and therapeutic implications.
Previous studies have reported that the presence of IR-A not only provides a high affinity receptor for IGF2 (8
), but also enhances the ability of insulin and IGF2 to signal through IGF1R (6
). Consistent with these earlier reports, we observed that physiological concentrations of IGF1, IGF2 or insulin activated the MAP kinase and Akt pathways (). These ligands also enhanced AML cell survival under serum-free conditions (). The effects of insulin were more pronounced in HL-60 cells, which expressed the highest amount of IR, whereas the effects of LR-3 and IGF-2 were higher in U937 cells, which express more IGF1R (, and data not shown).
Further studies demonstrated message for IGF1 and/or IGF2 in 70–90% of specimens from newly diagnosed AML patients (). While the present study was not designed to rule out a possible contribution to these signals from small numbers of contaminating stromal cells, the presence of IGF1 and/or IGF2 message in a variety of AML lines, including HL-60, K562, THP.1, KG1a and HEL ( and data not shown), indicates that myeloid cells can express these ligands. Coupled with recent reports that these ligands are secreted by clinical AML samples (28
), these results raise the possibility of an autocrine or paracrine pathway involving IGF1R and IR-A in AML. In view of the ability of IGF1R and IR-A ligands to activate downstream signaling (), such a pathway might contribute to the previously described constitutive activation of the MAP kinase (43
) and PI3K/Akt pathways (46
) in clinical AML specimens.
In further experiments, BMS-536924, an example of a new drug class that inhibits IGFR1R and IR almost equally (15
), diminished proliferation at concentrations as low as 200 nM () and induced apoptosis in susceptible AML lines (). Moreover, BMS-536924 inhibited colony formation in CD34+
clinical AML samples by as much as 98% (). There was, however, variability among clinical samples, as indicated by the fact that 10 µM BMS-536924 inhibited colony formation by 60–98% ().
This variability requires further investigation. No clear-cut correlation between different AML subtypes and BMS-536924 sensitivity is evident from the present studies, although the sample size was extremely small. On the other hand, we have observed differences in expression of some of the proteins that might be predicted to affect sensitivity, including IGF1R, IR, IRS1 and IRS2 ( and data not shown). Because expression of these polypeptides in the total blast population might not reflect levels in the cells that form colonies, it will be important to examine these polypeptides in specific AML subpopulations in the future. Other factors that might contribute to sensitivity differences include variability in expression of antiapoptotic Bcl-2 family members (15
) or the transporter ABCG2, which has recently been shown to efflux BMS-536924 (49
). Finally, it is conceivable that the ability or inability of cells to upregulate IGF1R and/or IR upon pathway inhibition might contribute to the variability. Future experiments will need to determine whether the BMS-536924-induced IGF1R upregulation observed in reflects increased gene expression, message stabilization, and/or posttranslational changes, whether similar effects are observed in clinical leukemia specimens, and whether this upregulation affects drug sensitivity.
Additional studies are also required to determine whether a dual IGF1R/IR inhibitor such as BMS-536924 exhibits selectivity for AML as compared to normal progenitors. It is known that primitive CD34+
normal progenitors lack IGF1R expression and are resistant to IGF1R downregulation (20
). Although it has been suggested that IR signaling might provide a survival signal for normal CD34+
), the paucity of marrow toxicity in mice treated with the IGF1R/IR inhibitor BMS-554417 (15
), coupled with the limited effects of NVP-AEW541 on normal human CD34+
) at concentrations that inhibit IR in addition to IGF1R, suggests that effects of dual IGF1R/IR inhibition on normal myeloid progenitors will be modest.
In summary, the present results not only demonstrate that IR-A, like IGF1R, is widely expressed in AML cell lines and clinical samples, but also show that the dual IGF1R/IR inhibitor BMS-536924 induces apoptosis in AML cell lines and profoundly inhibits colony formation in a subset of AML samples in vitro. Based on these results, further preclinical and possible clinical testing of therapies that simultaneously target IGF1R and IR in AML appear warranted.