The IL-3, IL-5, and GM-CSF common receptor beta chain mediates oncogenic activity of FLT3-ITD-positive AML.

FLT3-ITD is the most predominant mutation in AML being expressed in about one-third of AML patients and is associated with a poor prognosis. Efforts to better understand FLT3-ITD downstream signaling to possibly improve therapy response are needed. We have previously described FLT3-ITD-dependent phosphorylation of CSF2RB, the common receptor beta chain of IL-3, IL-5, and GM-CSF, and therefore examined its significance for FLT3-ITD-dependent oncogenic signaling and transformation. We discovered that FLT3-ITD directly binds to CSF2RB in AML cell lines and blasts isolated from AML patients. A knockdown of CSF2RB in FLT3-ITD positive AML cell lines as well as in a xenograft model decreased STAT5 phosphorylation, attenuated cell proliferation, and sensitized to FLT3 inhibition. Bone marrow from CSF2RB-deficient mice transfected with FLT3-ITD displayed decreased colony formation capacity and delayed disease onset together with increased survival upon transplantation into lethally irradiated mice. FLT3-ITD-dependent CSF2RB phosphorylation required phosphorylation of the FLT3 juxtamembrane domain at tyrosines 589 or 591, whereas the ITD insertion site and sequence were of no relevance. Our results demonstrate that CSF2RB participates in FLT3-ITD-dependent oncogenic signaling and transformation in vitro and in vivo. Thus, CSF2RB constitutes a rational treatment target in FLT3-ITD-positive AML.

Inhibition of wild-type and mutant Bcr-Abl by pyrido-pyrimidine-type small molecule kinase inhibitors.

Imatinib mesylate (STI571, Glivec), a 2-phenylaminopyrimidine small-molecule ATP competitor-type kinase inhibitor, proved to be active in Philadelphia-positive leukemias. Resistance toward imatinib develops frequently in advanced-stage Philadelphia-positive leukemia, and is even observed in chronic-phase chronic myelogenous leukemia. Point mutations within the BCR-ABL kinase domain emerged as a major mechanism of resistance toward imatinib. Mutations occur at positions that determine specific contacts of imatinib to the ATP-binding site. We aimed to examine whether pyrido-pyrimidine-type kinase inhibitors were capable of inhibiting both wild-type and mutant forms of BCR-ABL. We screened 13 different pyrido-pyrimidine with cells expressing wild-type and mutant BCR-ABL. All of the substances specifically suppressed the Bcr-Abl dependent phenotype and inhibited Bcr-Abl kinase activity with higher potency than imatinib. Two of the most active compounds were PD166326 and SKI DV-M016. Interestingly, these compounds suppressed the activation loop mutant Bcr-Abl H396P as effectively as wild-type Bcr-Abl. In addition, nucleotide-binding loop mutations (Y253H, E255K, and E255V) were selectively and potently inhibited. In contrast, T315I, a mutant located at a position that makes a direct contact with imatinib, was not affected. This observation is consistent with the hypothesis that unlike imatinib, pyrido-pyrimidine inhibitors bind Bcr-Abl regardless of the conformation of the activation loop. We conclude that pyrido-pyrimidine-type kinase inhibitors are active against different frequently observed kinase domain mutations of BCR-ABL that cause resistance toward imatinib. Resistance as a consequence of selection of mutant BCR-ABL by imatinib may be overcome using second-generation kinase inhibitors because of their higher potency and their ability to bind Bcr-Abl irrespective of the conformation of the activation loop.

Sequential inhibitor therapy in CML: in vitro simulation elucidates the pattern of resistance mutations after second- and third-line treatment.

PURPOSE: Dasatinib and nilotinib are active in imatinib-resistant chronic myelocytic leukemia (CML) and many patients undergo sequential treatment. We aimed at modeling sequential tyrosine kinase inhibitor (TKI) resistance in vitro to compare the sequences imatinib-nilotinib-dasatinib and imatinib-dasatinib-nilotinib. EXPERIMENTAL DESIGN: We designed an in vitro model for sequential TKI resistance in CML. Replicates of imatinib-resistant cell lines were treated with dasatinib or nilotinib. Second-line resistant replicates were exposed to third-line treatment. RESULTS: Growth of all replicates in all three lines of treatment was associated with T315I. However, T315I occurred with low abundance and did not increase during sequential treatment. Nilotinib second-line more often gave rise to sequential resistance compared with dasatinib due to pre-existing P-loop mutations, especially at suboptimal drug concentration. In contrast, mutations predisposing to dasatinib resistance such as F317C/V and V299L did not occur before dasatinib exposure. Nilotinib third-line did not overcome imatinib-dasatinib resistance due to pre-existing T315I or P-loop/V299L or P-loop/F317 exchanges. Dasatinib third-line suppressed imatinib-nilotinib-resistant replicates with residual sensitivity. CONCLUSIONS: Sequential acquisition of BCR-ABL drug resistance mutations in CML might be underestimated. Resistance to sequential TKI monotherapy in vitro more often was associated with stepwise acquisition of drug-specific compound mutations compared with T315I. Pre-existing mutations strongly limited the activity of both third-line treatments, and the activity of nilotinib second-line in vitro critically depended on drug concentration.

FMS-like tyrosine kinase 3-internal tandem duplication tyrosine kinase inhibitors display a nonoverlapping profile of resistance mutations in vitro.

FMS-like tyrosine kinase 3 (FLT3) inhibitors have shown activity in the treatment of acute myelogenous leukemia (AML). Secondary mutations in target kinases can cause clinical resistance to therapeutic kinase inhibition. We have previously shown that sensitivity toward tyrosine kinase inhibitors varies between different activating FLT3 mutations. We therefore intended to determine whether different FLT3 inhibitors would produce distinct profiles of secondary, FLT3 resistance mutations. Using a cell-based screening approach, we generated FLT3-internal tandem duplication (ITD)-expressing cell lines resistant to the FLT3 inhibitors SU5614, PKC412, and sorafenib. Interestingly, the profile of resistance mutations emerging with SU5614 was limited to exchanges in the second part of the kinase domain (TK2) with exchanges of D835 predominating. In contrast, PKC412 exclusively produced mutations within tyrosine kinase domain 1 (TK1) at position N676. A mutation at N676 recently has been reported in a case of PKC412-resistant AML. TK1 mutations exhibited a differential response to SU5614, sorafenib, and sunitinib but strongly impaired response to PKC412. TK2 exchanges identified with SU5614 were sensitive to PKC412, sunitinib, or sorafenib, with the exception of Y842D, which caused a strong resistance to sorafenib. Of note, sorafenib also produced a highly distinct profile of resistance mutations with no overlap to SU5614 or PKC412, including F691L in TK1 and exchanges at position Y842 of TK2. Thus, different FLT3 kinase inhibitors generate distinct, nonoverlapping resistance profiles. This is in contrast to Bcr-Abl kinase inhibitors such as imatinib, nilotinib, and dasatinib, which display overlapping resistance profiles. Therefore, combinations of FLT3 inhibitors may be useful to prevent FLT3 resistance mutations in the setting of FLT3-ITD-positive AML.

Bcr-Abl resistance screening predicts a limited spectrum of point mutations to be associated with clinical resistance to the Abl kinase inhibitor nilotinib (AMN107).

In advanced-phase chronic myeloid leukemia (CML), resistance to imatinib mesylate is associated with point mutations in the BCR-ABL kinase domain. A new generation of potent ABL kinase inhibitors is undergoing clinical evaluation. It is important to generate specific resistance profiles for each of these compounds, which could translate into combinatorial and sequential treatment strategies. Having characterized nilotinib (AMN107) against a large panel of imatinib mesylate-resistant Bcr-Abl mutants, we investigated which mutants might arise under nilotinib therapy using a cell-based resistance screen. In contrast to imatinib mesylate, resistance to nilotinib was associated with a limited spectrum of Bcr-Abl kinase mutations. Among these were mutations affecting the P-loop and T315I. Rarely emerging resistant colonies at a concentration of 400 nM nilotinib exclusively expressed the T315I mutation. With the exception of T315I, all of the mutations that were identified were effectively suppressed when the nilotinib concentration was increased to 2000 nM, which falls within the peak-trough range in plasma levels (3.6-1.7 microM) measured in patients treated with 400 mg twice daily. Our findings suggest that nilotinib might be superior to imatinib mesylate in terms of the development of resistance. However, our study indicates that clinical resistance to nilotinib may be associated with the predominant emergence of T315I.

A cell-based screen for resistance of Bcr-Abl-positive leukemia identifies the mutation pattern for PD166326, an alternative Abl kinase inhibitor.

In Philadelphia-positive (Ph(+)) leukemia, point mutations within the Bcr-Abl kinase domain emerged as a major mechanism of resistance to imatinib mesylate. We established a cell-based screening strategy for detection of clinically relevant point mutations using Bcr-Abl-transformed Ba/F3 cells. We identified 32 different single-point mutations within the kinase domain of Bcr-Abl. The pattern and frequency of mutations in this cell culture-based screen resembled the pattern and frequency observed in resistant patients. We then applied this screen to an alternative Abl kinase inhibitor. Using PD166326, the frequency of resistant colonies emerging at 5 to 10 times the median growth inhibition (IC50) of PD166326 was significantly lower than with imatinib. In addition, PD166326 produced a distinct pattern of Bcr-Abl mutations. The majority of mutations that came up with both imatinib and PD166326 could effectively be suppressed by increasing the dose of PD166326 to 50 to 500 nM. In contrast, only a few mutations could be suppressed by increasing the imatinib dose to 5 to 10 microM. However, 3 mutations affecting F317 displayed complete resistance to PD166326, but could be effectively inhibited by standard concentrations of imatinib. Thus, this robust and simple screening system provides a rational basis for combinatorial and sequential treatment strategies in targeted cancer therapy.