ALBERT

Description: Abstract 3247 Poster Board III-1 The tyrosine kinase inhibitor Imatinib is the gold standard in conventional treatment of CML. However, the emergence of resistance to IM remains a major problem. Alternative therapeutic strategies of IM-resistant BCR-ABL positive leukemias are urgently needed. One promising target for anticancer therapeutics is represented by the Aurora kinase family. These serine/threonine kinases are involved in regulating multiple steps of mitosis, including formation of bipolar spindle, chromosome alignment, spindle checkpoint function and cytokinesis. We report on studies accomplished with a small molecule inhibitor AS703569 (Merck Serono), which targets Bcr-Abl and Aurora kinases A-C. We could show that AS703569 exhibited strong anti-proliferative and pro-apoptotic activity against murine BaF3- cells ectopically expressing wild type (wt) or IM-resistant BCR-ABL mutants, including those harbouring the strongly resistant T315I mutation. This effect was observed already at rather low-AS703569 concentrations, at which Aurora- but not the Bcr-Abl kinase was inhibited. Furthermore, in cell cycle analysis we observed cells with a large 4N peak and DNA content more than 4N, indicating extensive polyploidisation, a consequence of continued cell cycle progression in the absence of cell division. Recent studies have revealed that this phenotype is based on suppression of Aurora B kinase activity, indicating that Aurora B inhibition is the major effect of AS703569 in Bcr-Abl positive cells. To confirm this assumption we designed MSCV based retroviruses encoding different point mutations in the Aurora B ATP binding site, which should lead to resistance against AS703569. By this strategy we were able to identify an AS703569 resistant mutant (Aurora B G216V). This mutant shows significant resistance in vitro and is able to augment the antiproliferative capacity of AS703569 in Bcr-Abl positive cells. Taken together, our data demonstrate that anti-proliferative effects of AS703569 in Bcr-Abl positive cells are primarily mediated by functional inhibition of Aurora kinases, especially of Aurora kinase B. Since Aurora kinases are clearly implicated in tumorgenesis, they will become a high potential therapeutic target for anticancer therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Clonal selection of cells harboring point mutations of the BCR-ABL kinase domain are considered a major cause of resistance to imatinib. More than 40 different point mutations have been described that cause a variable degree of imatinib resistance, and display a differential response to alternative kinase inhibitors, like dasatinib or nilotinib. Here, we describe three cases (2 m, 1 f) with imatinib resistant chronic myelogenous leukemia (CML) associated with a specific deletion of 81 bp of ABL exon 4. Patients were diagnosed with chronic phase (CP) CML at the age of 52, 54, and 68 years. After initial interferon alpha based therapies for 32, 60, and 71 mo, imatinib therapy was initiated at dosages between 400–800 mg per day. After 18, 24, and 29 mo patients lost hematologic response in CP CML (n=2) or progressed to lymphoid blast crisis (BC, n=1). Molecular analysis of the ABL kinase domain revealed a deletion of a 81 bp fragment associated with a loss of amino acids 248–274 in all cases. In one patient, an additional M351T mutation was found. In the two cases with CP CML, dasatinib was commenced for imatinib resistance, resulting in a partial hematologic and minor cytogenetic response (60 and 70% Ph+ metaphases, respectively) after 14 mo of therapy. The patient with lymphoid BC was treated with vincristine and prednisone and died 24 mo after appearance of imatinib resistance. In two cases, sequencing of genomic DNA revealed an underlying CTG/GTG mutation associated with a L248V amino acid switch. The point mutation activated a cryptic splice site within ABL exon 4 leading to an in-frame splice variant characterized by the loss of a 81 bp 3′ portion of exon 4. We sought to evaluate the BCR-ABL kinase activity of the splice variant and the response to tyrosine kinase inhibitors in vitro. The 81 bp deletion of p210 BCR-ABL was cloned using cDNA from one of the patients. Using this construct, retrovirally transduced Ba/F3 cells were transformed upon growth factor withdrawal. These cells expressed BCR-ABL at the transcript and protein levels. Presence of the 81 bp deletion was confirmed by sequencing. Despite the presence of the corresponding 27 amino acid P-loop deletion (Δ248–274), Western blot indicated strong autophosphorylation of BCR-ABL, which decreased in the presence of imatinib to non-detectable levels at concentrations of 1.25μM and above. In the presence of imatinib/nilotinib/dasatinib, the growth of BCR-ABL expressing Ba/F3 cells was shifted from an IC50 of 125/30/0.5nM for wild-type BCR-ABL to 470/185/1.9nM for Δ248–274 cells. Thus, in vitro data demonstrate that deletion of almost the entire P-loop does not abrogate BCR-ABL kinase activity and results in only marginal resistance towards ABL kinase inhibitors. We conclude that deletions of BCR-ABL may be the result of alternative splicing generated by point mutations associated with resistance to imatinib. The Δ248–274 splice variant retains BCR-ABL kinase activity and sensitivity to imatinib, nilotinib, and dasatinib.

Description: 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 μM) 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.

Description: Abstract 2912 Poster Board II-888 Constitutively activated variants of PDGFRA, PDGFRB can be found in a subset of patients with myeloid neoplasms associated with eosinophilia. The most common is FIP1L1-PDGFRA (FP). Patients with PDGFR-A and -B rearranged myeloproliferation respond to treatment with imatinib. However, single cases of clinical resistance due to a secondary FP/T674I mutation have been reported. In CML, more than 40 different exchanges have been described that confer imatinib resistance, and sequential treatment with imatinib and novel Abl kinase inhibitors has become reality. Nilotinib and sorafinib are potent alternative inhibitors of PDGFR-A and -B. We therefore hypothesized that available PDGFR kinase inhibitors might produce specific profiles of secondary FP kinase domain mutations mediating inhibitor resistance. To this aim, we selected clones of FP expressing Ba/F3 cells resistant to rising concentrations of imatinib, nilotinib, and sorafinib. In these, we identified 27 different PDGFRA kinase domain mutations. Imatinib, nilotinib and sorafenib produced distinct profiles of resistance mutations. During selection with imatinib, FP/T674I predominated with rising concentrations. FP/T674I corresponds to Bcr-Abl/T315I, which is frequently found in imatinib resistant CML. In contrast to imatinib, nilotinib and sorafenib produced a significantly lower frequency of resistant cell clones. Also, T674I disappeared at therapeutic nilotinib concentrations in favour of T674I+T874I and D842V. Sorafinib displayed a distinct profile of mutations including D842V, but not T674I. Following cloning and expression of all FP variants, dose-response analysis indicated that full cross-resistance to all three inhibitors was limited to D842V, whereas the gatekeeper T674I exchange retained sensitivity to sorafenib and nilotinib, and T674I+T874I was responsive to sorafenib only. In silico structure modelling indicated that differences in inhibitor response observed in distinct clusters of FP mutations identified in drug-resistant clones are based on differences of sorafenib versus imatinib and nilotinib in key drug - protein target interactions in PDGFR family kinases. Besides direct inhibitor binding effects, we propose that the identified exchanges shift an inactive-active conformation equilibrium and thereby affect binding of type II inhibitors like imatinib, nilotinib and sorafenib. Our results predict PDGFR variants that might come up in patients with myeloproliferation positive for PDGFR-A or -B fusions treated with imatinib, nilotinib or sorafenib. These findings will help in selection of an appropriate second line PDGFR kinase inhibitor when resistance to imatinib emerges, and will guide drug design. Moreover, our data can be translated to other neoplasms driven by activated forms of PDGFR-A or -B including GIST, CMML, and dermatofibrosarcoma protuberans. Disclosures: Off Label Use: Use of sorafenib and nilotinib in myeloproliferation with prominent eosinophilia.

Description: Constitutive activity of the Bcr-Abl kinase has been shown to be the causative event in the molecular pathogenesis of CML and Ph+ ALL. Even though Imatinib, a small molecule inhibitor of the Abl kinase, is very effective in treating the disease, development of resistance to this drug is a common phenomenon in advanced stage disease. Using Leptomycin B (LMB), a nuclear export blocker, Vigneri et al. have shown that Bcr-Abl kinase activity trapped in the nucleus induces cell death, thus being a new potential treatment option for Bcr-Abl expressing diseases. To test whether this approach might be effective in the case of imatinib resistant leukaemia several Bcr-Abl positive, imatinib-resistant Ba/F3 clones were established and characterized: 1. Ba/F3 cells resistant due to a point mutation in the Abl kinase domain at position T315I. 2. Ba/F3 cells resistant due to amplification of the Bcr-Abl kinase. 3. Ba/F3 cell clones resistant to imatinib with clear features of clonal evolution. All four cell lines were tested for the efficacy of a combined treatment approach with imatinib and leptomycin B. This combination was very effective in inducing apoptosis in imatinib resistant Ba/F3 cells displaying Bcr-Abl amplification. One out of two cell lines with signs of clonal evolution also responded to this combination treatment. However, imatinib resistant cells expressing the Bcr-AblT315I mutant kinase were completely resistant to this approach. Thus, these results are in agreement with the proposed hypothesis of Vigneri and wang that the combined effect of leptomycin B and imatinib requires Bcr-Abl kinase inhibition (which is not achieved in cells expressing Bcr-Abl T315I) by imatinib and nuclear entrapment by LMB To check the feasibility of this combined treatment for purging purposes, in vitro colony forming assays were performed with mixed cultures of Bcr-Abl positive BaF3 cells and primary murine bone marrow. All colonies formed after combined treatment with imatinib and LMB were derived from BM cells, indicating selective toxicity towards Bcr-Abl positive cells while sparing primary murine bone marrow cells. We are currently evaluating the effectivity of the combined LMB/imatinib treatment as purging strategy in a murine bone marrow transplantation model.

Description: Mutations causing resistance to therapeutic kinase inhibition can be identified in target kinases in various malignant diseases, such as Bcr-Abl in CML, FLT3-ITD in AML, cKit in GIST, EGFR in NSCLC, and FIP1L1-PDGFRalpha in HES/CEL. Thus, mutations in target kinases constitute a general mechanism of resistance to therapeutic kinase inhibiton. It has been shown that sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor. 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-ITD expressing cell lines resistant to the FLT3 inhibitors su5614 and PKC412. The frequency of resistant clones per million cells in the presence of inhibitor at 10 times the IC50 was 0.17 for su5614 (1,5?M) and 0 with PKC412 (100nM). When FLT3-ITD transformed cells were mutagenized with ENU (ethylnitrosourea) prior to addition of inhibitors, the frequency of resistant clones increased to 2.33 with su5614, and to 0.34 with PKC412. When we analyzed FLT3-ITD TK1 and TK2 for mutations that may confer inhibitor resistance, we found mutations in 127 out of 179 cell clones (71%) with su5614. Nine different exchanges affecting six positions in TK2 were identified, within or shortly behind the FLT3-ITD A-loop, with exchanges of D835 predominating. We did not detect exchanges affecting TK1. With PKC412, four out of 26 resistant clones (15%) contained mutations. In contrast to su5614, no mutations of TK2 were identified. However, three different amino acid exchanges emerging with PKC412 exclusively affected N676, which is located in TK1 of the FLT3-ITD split kinase domain. This is in line with the observation of a FLT3-ITD N676K exchange identified in a single AML patient with clinical resistance to PKC412 (Heidel et al, 2006). When expressed in cell lines, the identified TK2 A-loop mutations shifted dose-response curves to higher concentrations of su5614, but did not affect response to PKC412, su11248 or sorafenib. However, Y842D caused a strong resistance towards sorafenib, but not to su11248 or PKC412. Recent results also demonstrate a differential response of TK1 N676 exchanges towards different FLT3 inhibitors. We are currently analyzing su11248- and sorafenib- resistant FLT3-ITD clones for specific resistance mutations. Our results demonstrate that cell-based resistance screening is a useful and valid tool for prediction of resistance mutations to kinase inhibitors. In contrast to Bcr-Abl kinase inhibitors such as imatinib, nilotinib and dasatinib, which display highly overlapping resistance profiles, FLT3 kinase inhibitors may generate distinct, non-overlapping profiles. This opens the possibility of using combined FLT3 inhibitor therapy in AML to prevent resistance due to FLT3-ITD kinase domain mutations.

Description: In advanced-phase CML, resistance to imatinib mesylate is frequently associated with point mutations in the Bcr-Abl kinase domain. New, highly potent Abl kinase inhibitors such as AMN107 and BMS-354824, have recently entered clinical trials. Data from analyses of resistant patients will be available not before a large number of resistant patients will have been treated within clinical trials. Therefore, it will be important to generate specific resistance profiles for each compound prior to its therapeutic application. Using a cell-based screening method for resistance of Bcr-Abl positive leukemia to Abl kinase inhibitors, we generated a resistance profile for AMN107 and compared it to the resistance profile of imatinib mesylate. In contrast to imatinib, resistance to AMN107 was associated with a very limited spectrum of Bcr-Abl kinase mutations. While 26 exchanges at 21 positions occured with imatinib, the AMN107 screen revealed eight different exchanges at seven amino acid positions, with four exchanges affecting the P-loop. Novel mutations which have never been observed with imatinib, either in vitro or in resistant patients, emerged in the presence of AMN107 including an F359 exchange to isoleucine and a Q252H/S349L double mutant. In contrast to imatinib, the frequency of resistant colonies dramatically decreased with increasing AMN107 concentrations. Rarely emerging resistant colonies at a concentration of 400 nM AMN107 exclusively contained T315I. With the exception of T315I, all mutations that were identified were effectively suppressed when AMN107 was increased to 2000 nM, a concentration which is achieved in plasma in treated patients. Thus, in this system, increasing the AMN107 concentration to 400 nM prevented the emergence of resistant colonies, with the exception of T315I. Our findings suggest that AMN107 might be superior to imatinib in terms of the development of resistance. Also, AMN107 at clinically relevant concentrations may overcome imatinib resistant disease, including cases with expression of P-loop mutations. However, our study indicates that clinical resistance to AMN107 may be associated with the predominant emergence of T315I. Using this or similar approaches, it will be possible to provide information that translates into combinatorial and sequential treatment strategies and to determine critical plasma concentrations for mutations that might occur during treatment.

Description: FIP1L1-PDGFR alpha is a constitutively activated protein kinase which was reported in chronic eosinophilic leukemia (CEL) and in cases of hypereosinophilic syndrome and mastocytosis with eosinophilia. Imatinib is clinically active against FIP1L1-PDGFRA positive disease. However, clinical resistance to imatinib has been observed in FIP1L1-PDGFRA positive leukemia and was shown to occur due to a secondary mutation (T674I) in the PDGFR alpha kinase domain. Using a screening strategy to identify imatinib resistant mutations, we generated numerous imatinib resistant cell clones. Analysis of the PDGFRA kinase domain in these cell clones revealed a broad spectrum of resistance mutations including the clinically reported exchange T674I. Interestingly, one of the abundant mutations was a Phe to Ser exchange at position 604 (F604S), which occurred alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S in contrast to D842H and F604+D842H did not increase the biochemical or cellular IC50 value to imatinib when compared to wild-type (wt). However, F604S and F604S+D842H transformed Ba/F3, NIH3T3 and mouse bone marrow more efficiently compared to wt and D842H, respectively. Also, F604S and F604S+D842H showed strong activation of Stat5, ERK and Akt compared to wt and D842H. Immunoprecipitation and immunoblotting indicated increased amounts of FIP1L1-PDGFRA protein in F604S versus wt cells. Moreover, SRC coimmunoprecipitated with FIP1L1-PDGFRA in wt, but not F604S cells. We hypothesized that F604S might interfere with FIP1L1-PDGFRA protein stability, and that SRC might be involved in this process. GST pull down experiments using SRC-SH2 domain showed lesser binding of FIP1L1-PDGFRA/F604S compared to wt. Similarly, using a GST-PDGFRA fragment, more SRC was precipitated with wt compared to F604S. Importantly both, the SRC inhibitor PD166326 and SRC siRNA mimicked the F604S phenotype and resulted in stabilization of the wt protein. Also, co-expression of SRC in 293T cells augmented degradation of wt, but not F604S FIP1L1-PDGFRA, indicating that SRC is a negative regulator of FIP1L1-PDGFRA protein stability. Similar results were obtained with an exchange in near proximity to F604. Kinase-defective SRC had no effect on FIP1L1-PDGFRA stability, indicating that kinase activity of SRC is necessary for its effect on FIP1L1-PDGFRA stability. Moreover, kinase defective FIP1L1-PDGFRA (G610R) was not degraded indicating that kinase activity of FIP1l1-PDGFRA is necessary for its own degradation. Taken together, imatinib resistance screening in FIP1L1-PDGFRA identified a novel class of resistance mutations, that do not act by impeding drug binding to the target, but rather increase target protein levels by interfering with its SRC mediated degradation.