ALBERT

Description: Chronic myeloid leukemia (CML) is a stem cell disease characterized by the BCR/ABL oncoprotein. The ABL kinase inhibitor imatinib is effective in most patients and considered standard first line therapy. However, not all patients show a long-lasting response. Treatment failure is usually associated with the occurrence of imatinib-resistant mutants of BCR/ABL. For these patients, novel multi-kinase inhibitors such as dasatinib represent alternative treatment options. Still, however, not all patients respond to these drugs, especially when leukemic cells bear the BCR/ABL mutant T315I that confers resistance against most kinase-blockers. Bosutinib is a novel multi-kinase inhibitor that has been described to act growth-inhibitory in ABL-transformed leukemias. In the current study, we examined the effects of bosutinib alone and in combination with dasatinib on growth and survival of CML cells. Bosutinib was found to inhibit 3H-thymidine uptake and thus proliferation in imatinib-sensitive and imatinib-resistant K562 cells in a dose-dependent manner, with identical IC50 values (10–100 nM). Moreover, bosutinib was found to inhibit the growth of primary CML cells and Ba/F3 cells bearing various imatinibresistant mutants of BCR/ABL, except the T315I mutant (IC50〉1 μM). The growth-inhibitory effects of bosutinib were found to be associated with signs of apoptosis. Dasatinib showed similar effects on CML cells, and again did not block the growth of subclones bearing BCR/ABL T315I. Unexpectedly, however, we found that bosutinib and dasatinib synergize with each other in producing growth inhibition in primary CML cells exhibiting BCR/ABL T315I at pharmacologic concentrations (0.01–1 μM). Clear synergistic effects were also observed in imatinib-sensitive and imatinib-resistant K562 cells as well as in Ba/F3 cells bearing BCR/ABL T315I. In parallel, we performed multiplexed kinase assays as well as chemical proteomics analysis and mass spectrometry using K562 cells and primary CML cells and coupleable dasatinib and bosutinib analogues. In these experiments, dasatinib and bosutinib were found to express an overlapping, but non-identical profile of target kinases. As expected, both drugs were found to bind to wt ABL, SRC kinases, and TEC-family kinases including BTK. Specific targets preferentially bound and inhibited by bosutinib were STE20s, the FES/FER family, CAMKIIG, PYK2 and TBK1. We were also able to confirm that the dasatinib-targets KIT and PDGFRA are not recognized by bosutinib. Interestingly, whereas wt ABL (IC50

Description: Resistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). We recently have identified the heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) as a BCR/ABL-dependent survival molecule in CML cells. We here show that silencing Hsp32/HO-1 in CML cells by an siRNA approach results in induction of apoptosis. Moreover, targeting Hsp32/HO-1 by either pegylated zinc protoporphyrine (PEG-ZnPP) or styrene maleic acid-micelle–encapsulated ZnPP (SMA-ZnPP) resulted in growth inhibition of BCR/ABL-transformed cells. The effects of PEG-ZnPP and SMA-ZnPP were demonstrable in Ba/F3 cells carrying various imatinib-resistant mutants of BCR/ABL, including the T315I mutant, which exhibits resistance against all clinically available BCR/ABL tyrosine kinase inhibitors. Growth-inhibitory effects of PEG-ZnPP and SMA-ZnPP also were observed in the CML-derived human cell lines K562 and KU812 as well as in primary leukemic cells obtained from patients with freshly diagnosed CML or imatinib-resistant CML. Finally, Hsp32/HO-1–targeting compounds were found to synergize with either imatinib or nilotinib in producing growth inhibition in imatinib-resistant K562 cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In summary, these data show that HO-1 is a promising novel target in imatinib-resistant CML.

Description: Systemic mastocytosis (SM) is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC) in various internal organs. In most patients, the D816V-mutated variant of c-KIT, which mediates resistance against several tyrosine kinase (TK) inhibitors like imatinib, is found. In advanced SM, the response of neoplastic MC to conventional drugs is poor and the prognosis is grave. Therefore current research is attempting to identify novel targets in neoplastic MC. Polo-like kinase 1 (Plk-1) is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias. In the present study, we analyzed expression and function of Plk-1 in neoplastic human MC, and asked whether Plk-1 can serve as a target of therapy in SM. As determined by immunohistochemistry, primary neoplastic MC were found to display activated/phosphorylated Plk-1 in all patients examined (n=5). The human MC leukemia cell line HMC-1 was also found to exhibit activated Plk-1. In addition, we found that primary neoplastic MC as well as HMC-1 cells express Plk-1 mRNA in RT-PCR experiments. As assessed by 3H-thymidine-uptake experiments, the Plk-1-targeting drug BI 2536 (Boehringer Ingelheim GmbH, Germany) was found to inhibit the proliferation of HMC-1 cells in a dose-dependent manner (IC50 5–15 nM). The effect of BI 2536 was seen in both subclones of HMC-1, i.e. in HMC-1.1 cells displaying KIT G560V (but not KIT D816V), and HMC-1.2 cells exhibiting both KIT G560V and KIT D816V, with comparable IC50 values. Moreover, BI 2536 was found to inhibit the proliferation of primary neoplastic cells, with IC50 values ranging between 5 and 50 nM. The growth-inhibitory effects of BI 2536 on HMC-1 cells were found to be associated with mitotic arrest and G2-M cell cycle arrest as well as consecutive apoptosis. In normal bone marrow or peripheral blood mononuclear cells, neither mitotic cell arrest nor apoptosis were observed after treatment with BI 2536. In a consecutive phase of the study, we asked whether combined targeting of KIT D816V and Plk-1 would lead to synergistic drug-interactions. For this purpose, HMC-1 cells and primary neoplastic MC were coincubated with BI 2536 and midostaurin (PKC412), a multitargeted kinase inhibitor that blocks KIT D816V TK activity. In these experiments, BI 2536 was found to synergize with midostaurin in counteracting the proliferation of HMC-1 cells and primary neoplastic MC. In conclusion, our data show that activated Plk-1 is detectable in MC neoplasms and plays a role in cell cycle progression and viability of neoplastic MC. Targeting of Plk-1 with BI 2536 leads to growth inhibition and apoptosis in neoplastic MC. Furthermore, BI 2536 synergizes with midostaurin in counteracting growth of neoplastic MC. Targeting of Plk-1 may be an attractive new pharmacologic concept in advanced SM.

Description: Abstract 3382 Siglec-3 (CD33) is an established therapeutic target in acute myeloid leukemia (AML). We and others have shown that CD33 is expressed on immature CD34+/CD38- stem cells in AML. We here report that leukemic stem cells obtained from patients with chronic myeloid leukemia (CML) display high levels of CD33, and that CD33 may serve as a potential target in CML. As assessed by multi-color flow cytometry, CD34+/CD38-/CD123+ CML progenitor cells in chronic phase (CP) were found to express significantly higher levels of CD33 compared to normal CD34+/CD38- bone marrow stem cells (figure). By contrast, similar levels of the cell surface antigen MDR1 (CD243) were detected when comparing normal and CML progenitors. In chronic phase (CP) CML, CD33 was found to be expressed homogeneously on most or all CD34+/CD38- stem cells. In patients with accelerated (AP) or myeloid blast phase (BP), CML stem cells also co-expressed CD33, but the levels of CD33 varied from donor to donor, and in one patient, most CML stem cells appeared to be CD33-negative cells. In two patients with CML, CD34+/CD38- cells were highly enriched by cell sorting (purity 〉98%) and found to contain CD33 mRNA in qPCR analysis. The presence of BCR/ABL and thus the leukemic origin of these cells was confirmed by FISH analysis and PCR. We then examined the effects of the CD33-targeted drug gemtuzumab/ozogamicin (GO) on growth of primary CML cells. As assessed by 3H-thymidine uptake, GO produced growth inhibition in leukemic cells in all patients tested (CP, n=13; AP, n=3). The effects of GO on leukemic cell growth were dose-dependent and occurred at relatively low concentrations, with IC50 values ranging between 1 and 100 ng/ml. GO effects were also seen in precursor-enriched Lin-negative CML cells (n=3). As assessed by Annexin-V staining, GO was found to induce apoptosis in CD34+/CD38- CML progenitor cells. Next we investigated drug combination effects. In these experiments, GO was found to synergize with nilotinib and bosutinib in producing growth inhibition in primary CML cells. In conclusion, CD33 is expressed abundantly on immature CD34+/CD38- progenitor cells in CML. Whether GO can be employed to eradicate residual leukemic stem cells in CML patients alone or in combination with BCR/ABL kinase inhibitors remains at present unknown. Figure: Expression of CD33 on CD34+/CD38- cells in normal bone marrow (n=7) and patients with CML in chronic phase (CP, n=16) or advanced phase (AP/BP, n=11) of the disease. Figure:. Expression of CD33 on CD34+/CD38- cells in normal bone marrow (n=7) and patients with CML in chronic phase (CP, n=16) or advanced phase (AP/BP, n=11) of the disease. Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Description: Abstract 3394 Resistance to imatinib is a major clinical problem and challenge in advanced chronic myeloid leukemia (CML). In most patients, drug-resistant mutants of BCR/ABL are detectable. Although most of these mutants still are responsive to second generation BCR/ABL kinase inhibitors (KI) such as nilotinib or dasatinib, drug responses are often short-lived. The BCR/ABL mutant T315I confers resistance against all available BCR/ABL KI, including nilotinib and dasatinib. More recent data suggest that several Aurora kinase (AuK) inhibitors block the kinase activity of BCR/ABL T315I. We have examined the growth-inhibitory effects of the AuK/ABL inhibitor R763/AS703569 (Merck-Serono, Darmstadt, Germany) on primary CML cells (chronic phase, n=12), the CML cell line K562, and Ba/F3 cells transfected with various imatinib-resistant mutants of BCR/ABL. As assessed by 3H-thymidine-uptake, R763/AS703569 was found to inhibit proliferation in imatinib-sensitive and imatinib-resistant primary CML cells in all donors tested, in imatinib-resistant and imatinib-responsive K562 cells, and in Ba/F3 cells harbouring various mutants of BCR/ABL (E255K, Y253F, H396P, T315I). The effects of R763/AS703569 on BCR/ABL-transformed cells were dose-dependent with IC50 values ranging between 0.001–0.1 μ M in K562 cells,

Description: Abstract 2280 In most patients with chronic myeloid leukemia (CML), complete cytogenetic remission can be achieved with the BCR/ABL tyrosine kinase inhibitor (TKI) imatinib. However, not all patients are long-term responders. A major cause of acquired resistance against imatinib is the development of BCR/ABL mutations in subclones. In most of these patients, a second generation TKI is prescribed. However, the T315I mutant of BCR/ABL introduces resistance against most TKI, including nilotinib and dasatinib. One approach to overcome drug resistance in BCR/ABL T315I+ CML cells may be to apply drug combinations. Recent data suggest that the mechanisms through which dasatinib and nilotinib act on BCR/ABL differ from each other and that both drugs act on multiple additional targets in CML cells. Here, we show that dasatinib and nilotinib cooperate with each other in producing growth inhibition in imatinib-sensitive and imatinib-resistant CML cells, including subclones bearing BCR/ABL T315I. The drug combination was tested on leukemic cells obtained from 9 patients with chronic phase (CP) CML and 3 with blast phase (PB) of CML. Samples were assessed from 4 patients at the time of diagnosis, and against cells from 8 patients (CP, n=5; BP, n=3) who had developed resistance against one or more BCR/ABL TKI. In all 3 patients in PB, the T315I mutant was detectable. As expected, nilotinib and dasatinib failed to inhibit proliferation of cells harbouring BCR/ABL T315I when applied as single agents. However, the combination xnilotinib+dasatinibx produced synergistic effects in most samples, including primary CML cells and Ba/F3 cells harbouring BCR/ABL T315I. Interestingly, in all 3 patients with BP (BCR/ABL T315I+), strong cooperative or even synergistic growth-inhibitory effects were observed in primary CML cells, resulting in substantial anti-leukemic effects seen at reasonable (pharmacologic) drug concentrations (〈 1 μ M) (figure). Based on these results, we treated one patient with TKI-resistant CML in hematologic relapse in whom 2 BCR/ABL mutant-bearing subclones, one clinically resistant against nilotinib (F359V) and one clinically resistant against dasatinib (F317L) had been detected, with a combination of nilotinb (800 mg p.o. daily) and dasatinib (50 mg/day p.o., days 1–5 every third week). A transient hematologic response was obtained in this patient, and except for mild bone pain, no side effects were recorded. Moreover, we were able to show that during treatment with xnilotinib+dasatinibx, the number of CD34+/CD38-/CD33+ CML stem cells decreased from clearly measurable levels (0.005%) to nearly undetectable levels (0.0002%). Finally, ex vivo analyses of leukemic blood cells confirmed, that the combination xnilotinib+dasatinibx produced strong cooperative growth-inhibitory effects in both disease-components, i.e. the F359V-bearing subclone and the F317L-bearing subclone. In summary, our data show that the combination of dasatinib and nilotinib can override acquired TKI resistance in CML, and can suppress growth of various imatinib-resistant subclones including cells that bear BCR/ABL T315I or other BCR/ABL mutants. Whether this combination can suppress imatinib-resistant subclones in CML for prolonged time periods or even can eradicate neoplastic stem cells remains in CML patients to be determined. Synergistic effects of nilotinib and dasatinib on primary leukemic cells obtained from a patient with a BCR/ABL T315I+ blast phase of CML Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Description: Abstract 3972 Systemic mastocytosis (SM) is a myeloid neoplasm defined by abnormal growth and accumulation of neoplastic mast cells (MC) in one or more internal organs. In most patients, the D816V-mutated variant of KIT is detectable. This mutant supposedly confers resistance against several tyrosine kinase inhibitors including imatinib and masitinib. In aggressive SM (ASM) or mast cell leukemia (MCL) the response to conventional drugs is poor and the prognosis is grave. In these patients, additional KIT-independent signalling pathways and molecules, such as BTK and LYN may play an important role in disease evolution and MC proliferation. R763/AS703569 is a multikinase inhibitor that blocks the kinase activity of KIT, BTK, LYN, Aurora-Kinase-A, Aurora-Kinase-B, ABL, AKT, and FLT3. We analyzed the effects of R763/AS703569 on growth and survival of the human mast cell leukemia cell line HMC-1 and the canine mastocytoma cell line C2. Two subclones of HMC-1 were used, one expressing KIT D816V (HMC-1.2) and one lacking KIT D816V (HMC-1.1). Both HMC-1 subclones were found to express Aurora-Kinase-A mRNA and Aurora-Kinase-B mRNA in RT-PCR experiments. As assessed by 3H-thymidine uptake, R763/AS703569 was found to inhibit proliferation of HMC-1 cells in a dose-dependent manner, with lower IC50 values obtained in HMC-1.2 cells (1-5 nM) compared to HMC-1.1 cells (10-10-50 nM). Moreover, R763/AS703569 produced growth inhibition in C2 cells (IC50: 1–5 nM). As assessed by light microscopy and Tunel assay, the growth-inhibitory effects of R763/AS703569 were found to be accompanied by apoptosis in all three cell lines. Correspondingly, R763/AS703569 was found to induce cleavage of caspase-3, caspase-8, and caspase-9 in HMC-1 cells. Moreover, R763/AS703569 was found to induce a G2/M cell cycle arrest in HMC-1 cells and C2 cells after 24 hours. In order to define the target spectrum for R763/AS703569 in HMC-1 cells, Western blot experiments were performed. In these experiments, R763/AS703569 was found to inhibit the phosphorylation of KIT, Aurora-Kinase-A, and BTK in HMC-1.1 cells, whereas no effects of R763/AS703569 on phosphorylation of LYN were seen. We then combined R763/AS703569 with dasatinib, a drug known to block LYN activation in HMC-1 cells. In these experiments, we were able to show that both drugs cooperate with each other in inducing apoptosis in HMC-1.1 cells and HMC-1.2 cells. In summary, our data suggest that R763/AS703569 is a novel promising drug that should be tested for its anti-neoplastic effects in patients with ASM and MCL in clinical trials. Complete inhibition of growth of neoplastic MC may require drug combinations employing R763/AS703569 and other targeted or cytotoxic drugs. Disclosures: Sarno: Merck-Serono: Employment. Valent:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Merck-Serono: Research Funding.

Description: Heme oxygenase 1 (HO-1), also known as heat shock protein 32 (Hsp32), has recently been identified as a stress-related survival molecule that acts anti-apoptotic and cytoprotective in inflammatory reactions. Recent data suggest that HO-1/Hsp32 is also expressed in neoplastic cells in various malignancies. In the present study, we provide evidence that HO-1 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML, n=17) and in various AML cell lines such as HL60, KG1, KG1a, and U937. Expression of HO-1 mRNA was demonstrable by RT-PCR, and the HO-1 protein by immunocytochemistry and Western blotting. In addition, we were able to demonstrate expression of HO-1 mRNA and of HO-1 protein in the CD34+/CD38− progenitor/stem cell fraction in the leukemic clone in patients with AML. The HO-1 inductor hemin (10 μM) was found to promote expression of HO-1 in AML cells. Incubation with the HO-1-targeting drugs pegylated zink protoporphyrin (PEG-ZnPP) or styrene maleic acid-conjugated ZnPP (SMA-ZnPP), resulted in a dose-dependent inhibition of growth of leukemic cells at pharmacologic concentrations (IC50: 5–20 μM for cell lines and primary AML cells). The SMA-ZnPP-induced growth-inhibition of AML cells were found to be associated with induction of apoptosis as evidenced by light microscopy, electron microscopy, and by a Tunel assay. In consecutive experiments, combination experiments were performed using SMA-ZnPP and AML cell lines. In these experiments, SMA-ZnPP was found to synergize with cytarabine in producing growth inhibition in all AML cell lines tested. In summary, these data show that HO-1/Hsp32 is a novel survival factor and interesting target in AML. The clinical significance of this observation remains to be determined in forthcoming trials.

Description: The signal transducer and activator of transcription 5 (STAT5) has recently been implicated as essential pro-oncogenic factor in the pathogenesis of myeloid leukemias in mice (Cancer Cell2005;7:87–99). More recently, STAT5 activation has also been described to occur in human leukemias. However, so far, little is known about the expression of activated/tyrosine phosphorylated STAT5 (pSTAT5) in various myeloid neoplasms and about the distribution of pSTAT5 in the cellular compartments of the normal and leukemic bone marrow (bm). We have examined the expression of pSTAT5 in the bm in patients with acute myeloid leukemia (AML, FAB M0, n=3, M1, n=6, M2, n=4, M3, n=5, M4, n=5, M5, n=4, M6, n=5, M7, n=4), chronic myeloid leukemia (CML, chronic phase, n=4, accelerated phase, n=5, blast phase, n=5), and systemic mastocytosis (SM, n=30), as well as in the normal bm (n=5). Expression of pSTAT5 was determined on paraffin-embedded bm sections by immunohistochemistry using the pSTAT5-specific antibody AX1. In the normal bm, the antibody AX1 was found to react with megakaryocytes and immature myeloid progenitor cells, whereas erythroid cells and mature granulocytic cells did not stain positive for AX1. In patients with AML and CML, the distribution of pSTAT5 showed a similar pattern. In fact, pSTAT5 was found to be expressed in leukemic blast cells without differences among FAB types as well as megakaryocytic cells, but not in erythroid cells. In patients with SM, neoplastic mast cells were found to be immunoreactive for pSTAT5. Interestingly, in all patients and all cells examined, pSTAT5 was found to be localized in the cytoplasm rather than in the nucleus. The cytoplasmic distribution of pSTAT5 in neoplastic cells was confirmed by immunocytochemical staining experiments performed on primary isolated neoplastic cells (AML, CML, mastocytosis) and respective cell lines (U937, KG1, K562, KU812, HMC-1). In each case, the reactivity of neoplastic cells with the AX1 antibody was abrogated by preincubation of the antibody with a pSTAT5-specific blocking peptide. Moreover, the expression of cytoplasmic pSTAT5 in the leukemic cell lines was demonstrable by flow cytometry. To study the molecular mechanisms underlying STAT5-activation in neoplastic cells, Ba/F3 cells with doxycycline-inducible expression of disease-specific oncoproteins, namely BCR/ABL (CML) and KIT-D816V (SM) were employed. Induction of these oncoproteins in Ba/F3 cells resulted in massive activation of pSTAT5 and DNA binding activity as shown by EMSA and supershift assays. In summary, our data show that neoplastic cells in AML, CML, and SM express cytoplasmic pSTAT5, and that disease-related oncoproteins contribute to STAT5-activation. The particular cytoplasmic localization of pSTAT5 in neoplastic cells suggests that apart from its function as a transcription factor, pSTAT5 may have an additional role as a cytoplasmic regulator in these malignancies.