nitrophenols and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Both germline polymorphisms and tumor-specific genetic alterations can determine the response of a cancer to a given therapy. We previously reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), as well as other cancers [1]. The deletion polymorphism favored the generation of BIM splice forms lacking the pro-apoptotic BH3 domain, conferring a relative resistance to the TKI imatinib (IM). However, CML patients with the BIM deletion polymorphism developed both partial and complete IM resistance. To understand the mechanisms underlying the latter, we grew CML cells either with or without the BIM deletion polymorphism in increasing IM concentrations. Under these conditions, the BIM deletion polymorphism enhanced the emergence of populations with complete IM resistance, mimicking the situation in patients. Importantly, the combined use of TKIs with the BH3 mimetic ABT-737 overcame the BCR-ABL1-dependent and -independent resistance mechanisms found in these cells. Our results illustrate the interplay between germline and acquired genetic factors in confering TKI resistance, and suggest a therapeutic strategy for patients with complete TKI resistance associated with the BIM deletion polymorphism.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Biphenyl Compounds; Cell Line, Tumor; Dasatinib; Fusion Proteins, bcr-abl; Gene Deletion; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Proteins; Nitrophenols; Piperazines; Polymorphism, Genetic; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Pyrimidines; Sulfonamides

The Bcr-Abl tyrosine kinase regulates several Bcl-2 family proteins that confer resistance to apoptosis in chronic myeloid leukemia (CML) cells. Given p53's ability to modulate the expression and activity of Bcl-2 family members, we hypothesized that targeting Bcr-Abl, Bcl-2, and p53 concomitantly could have therapeutic benefits in blast crisis (BC) CML and in quiescent CML CD34+ cells that are insensitive to tyrosine kinase inhibitors (TKI). We examined the effects of the MDM2 inhibitor nutlin3a and its combination with the dual Bcl-2 and Bcl-xL inhibitor ABT-737, and the Bcr-Abl inhibitor nilotinib on BC CML patient samples. We found that in quiescent CD34+ progenitors, p53 expression is significantly lower, and MDM2 is higher, compared to their proliferating counterparts. Treatment with nutlin3a induced apoptosis in bulk and CD34+CD38- cells, and in both proliferating and quiescent CD34+ progenitor CML cells. Nutlin3a synergized with ABT-737 and nilotinib, in part by inducing pro-apoptotic, and suppressing anti-apoptotic, Bcl-2 proteins. Nilotinib inhibited the expression of Bcl-xL and Mcl-1 in BC CML cells. These results demonstrate that p53 activation by MDM2 blockade can sensitize BC CML cells, including quiescent CD34+ cells, to Bcl-2 inhibitor- and TKI-induced apoptosis. This novel strategy could be useful in the therapy of BC CML.

Topics: ADP-ribosyl Cyclase 1; Antigens, CD34; Antineoplastic Agents; Apoptosis; bcl-X Protein; Biphenyl Compounds; Blast Crisis; Cell Proliferation; Enzyme Activation; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Humans; Imatinib Mesylate; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Glycoproteins; Nitrophenols; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Pyrimidines; Sulfonamides; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Despite being highly effective for newly diagnosed chronic myeloid leukemia (CML), imatinib not only is inactive against quiescent CML stem cells, but also has limited activity against blast crisis (BC) CML. The relative activity of Bcr-Abl and the expression levels of antiapoptotic proteins in proliferating and quiescent CD34(+) BC CML progenitor cells and the effects of targeting antiapoptotic proteins in these cells are unknown. Here we report higher levels of p-CrkL in quiescent than in proliferating CD34(+) progenitor cells and comparable expression levels of Bcl-2, Bcl-xL, Mcl-1 and XIAP in the two populations in BC CML. Inhibition of Bcl-2/Bcl-xL by ABT-737 in cells from patients with tyrosine kinase inhibitor (TKI)-resistant BC CML promoted apoptosis in quiescent CD34(+) progenitor cells with an efficacy similar to that in proliferating cells. Combination of ABT-737 with imatinib (which decreases Mcl-1 levels) or triptolide (which decreases Mcl-1 and XIAP) synergistically induced death of both proliferating and quiescent CD34(+) progenitor cells obtained from TKI-resistant BC CML patients. These results suggest that antiapoptotic proteins are critical targets in BC CML and that activation of apoptosis signaling can eliminate both proliferating and quiescent CD34(+) progenitor cells in BC CML, independent of response to TKIs.

Topics: Antigens, CD34; Apoptosis; Benzamides; Biphenyl Compounds; Blast Crisis; Cell Line, Tumor; Diterpenes; Epoxy Compounds; Fusion Proteins, bcr-abl; Hematopoietic Stem Cells; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nitrophenols; Phenanthrenes; Piperazines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; RNA, Messenger; Signal Transduction; Sulfonamides

Chronic myeloid leukemia (CML) tumorigenicity is driven by the oncogenic BCR-ABL tyrosine kinase. Specific tyrosine kinase inhibitors (TKI) have been designed and are now used for the treatment of CML. These TKI induce apoptosis in leukemic cells in a BIM-dependent mechanism. We hypothesized that an increase in BIM activity could sensitize CML cells to TKI. We blocked the anti-apoptotic proteins of the Bcl-2 family by using ABT-737, a Bcl-2 and Bcl-XL inhibitor. ABT-737 modified Bcl-2 protein interactions toward a pro-apoptotic phenotype. Its combination with TKI resulted in a strong synergism in CML cell lines. The association also induced a large decrease in X-linked inhibitor of apoptosis (XIAP), followed by caspase-3 activation. This XIAP decrease was due to post-translational events. The mitochondrial serine protease HtrA2/Omi was identified as being responsible for this off-target effect. Then, ABT-737 and TKI cooperate at several levels to induce apoptosis of CML cells lines, and the benefit of this association was also observed in CML hematopoietic progenitors. Interestingly, a lethal effect was also observed in the more immature CD34(+)CD38(-) TKI-insensitive population. Combination therapy might by an interesting strategy for the treatment of CML patients.

Topics: Antigens, CD34; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Benzamides; Biphenyl Compounds; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; High-Temperature Requirement A Serine Peptidase 2; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Proteins; Mitochondrial Proteins; Neoplasm Proteins; Neoplastic Stem Cells; Nitrophenols; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Serine Endopeptidases; Sulfonamides; X-Linked Inhibitor of Apoptosis Protein

Chronic myeloid leukemia is the first disease in which the potential of molecular targeted therapy with tyrosine kinase inhibitors (TKIs) was realized. Despite this success, a proportion of patients, particularly with advanced disease, are, or become, resistant to this treatment. Overcoming resistance and uncovering the underlying mechanisms is vital for further improvement of clinical outcomes. Here we report the identification, development, and characterization of a novel chronic myeloid leukemia cell line carrying the additional chromosomal aberration t(3;12)(q26;p13) resulting in expression of the TEL/MDS1/EVI1 fusion protein, which is resistant to TKIs. Resistance to TKIs was overcome by the co-administration of the BH3-mimetic, ABT-737. In addition, application of EVI1-specific small interfering RNA decreased expression of the TEL/MDS1/EVI1 fusion, reduced resistance to imatinib, and increased sensitivity to ABT-737. Subsequent studies revealed a role for the BH3-only protein BAD, probably via a phosphoinositide 3-kinase/AKT-dependent pathway, as pharmacological inhibition of AKT could also resensitize cells to death from TKIs. These findings indicate a novel pathway of TKI resistance regulated by EVI1 proteins and provide a promising means for overcoming resistance in chronic myeloid leukemia and other hematological malignancies displaying EVI1 overexpression.

Topics: Adult; Antineoplastic Agents; Apoptosis; bcl-Associated Death Protein; Benzamides; Biphenyl Compounds; Cell Line, Tumor; Chromosomes, Human, Pair 12; Chromosomes, Human, Pair 3; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; ETS Translocation Variant 6 Protein; Female; Humans; Imatinib Mesylate; Immunoblotting; K562 Cells; Karyotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MDS1 and EVI1 Complex Locus Protein; Nitrophenols; Oncogene Proteins, Fusion; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-ets; Proto-Oncogenes; Pyrimidines; Repressor Proteins; RNA Interference; Sulfonamides; Transcription Factors; Translocation, Genetic

Although the BCR/ABL tyrosine kinase inhibitor imatinib is highly effective for treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia, relapse with emerging imatinib resistance mutations in the BCR/ABL kinase domain poses a significant problem. Here, we show that the multikinase inhibitor sorafenib inhibits proliferation and induces apoptosis at much lower concentrations in Ton.B210 cells when driven by inducibly expressed BCR/ABL than when driven by interleukin-3. The increased sensitivity to sorafenib was also observed in cells inducibly expressing BCR/ABL with the imatinib-resistant E255K or T315I mutation. Sorafenib-induced apoptosis in these cells and Ph+ leukemic cells was synergistically enhanced by rottlerin, bortezomib, or ABT-737 and inhibited by the pan-caspase inhibitor BOC-d-fmk or the overexpression of Bcl-XL. It was further revealed that sorafenib activates Bax and caspase-3 and reduces mitochondrial membrane potential specifically in BCR/ABL-driven cells. Sorafenib also inhibited BCR/ABL-induced tyrosine phosphorylation of its cellular substrates and its autophosphorylation in Ton.B210. It was finally shown that sorafenib inhibits the kinase activity of BCR/ABL as well as its E255K and T315I mutants in in vitro kinase assays. These results indicate that sorafenib induces apoptosis of BCR/ABL-expressing cells, at least partly, by inhibiting BCR/ABL to activate the mitochondria-mediated apoptotic pathway. Thus, sorafenib may provide an effective therapeutic measure to treat Ph+ leukemias, particularly those expressing the T315I mutant, which is totally resistant to imatinib and the second generation BCR/ABL inhibitors.

Topics: Acetophenones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Benzenesulfonates; Benzopyrans; Biphenyl Compounds; Boronic Acids; Bortezomib; Caspases; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Interleukin-3; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mitochondria; Mutation; Niacinamide; Nitrophenols; Phenylurea Compounds; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazines; Pyridines; Pyrimidines; Sorafenib; Sulfonamides

The effect of ABT-737, a BH3-mimicking inhibitor for anti-apoptotic Bcl-2 and Bcl-X(L), but not Mcl-1, against Bcr-Abl-positive (Bcr-Abl(+)) leukaemic cells was examined. ABT-737 potently induced apoptosis in Bcr-Abl(+) chronic myeloid leukaemia (CML) cell lines and primary CML samples in vitro and prolonged the survival of mice xenografted with BV173 cells, a CML cell line. Higher expression of anti-apoptotic Bcl-2 proteins reduced cell killing by ABT-737 in each cell line, but there was no correlation between the sensitivities to ABT-737 and the specific expression patterns of Bcl-2 family proteins among cell lines. Thus, the cell killing effect of ABT-737 must be determined not only by the expression patterns of Bcl-2 family proteins but also by other mechanisms, such as high expression of Bcr-Abl, or a drug-efflux pump, in CML cells. ABT-737 augmented the cell killing effect of imatinib in Bcr-Abl(+) cells with diverse drug-resistance mechanisms unless leukaemic cells harboured imatinib-insensitive Abl kinase domain mutations, such as T315I. The combination of homoharringtonine that reduces Mcl-1 enhanced the killing by ABT-737 strongly in Bcr-Abl(+) cells even with T315I mutation. These results suggest that ABT-737 is a useful component of chemotherapies for CML with diverse drug-resistance mechanisms.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Harringtonines; Homoharringtonine; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Survival Analysis; Transplantation, Heterologous; Tumor Cells, Cultured

Bcr-Abl is the cause of Philadelphia-positive (Ph(+)) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph(+) leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-X(L), greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph(+) leukemic cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Benzoquinones; Biphenyl Compounds; Boronic Acids; Bortezomib; Cell Line, Transformed; Cell Line, Tumor; Humans; Imatinib Mesylate; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-bcr; Pyrazines; Pyrimidines; Sulfonamides

Cell killing is a critical pharmacological activity of imatinib to eradicate Bcr/Abl+ leukemias. We found that imatinib kills Bcr/Abl+ leukemic cells by triggering the Bcl-2-regulated apoptotic pathway. Imatinib activated several proapoptotic BH3-only proteins: bim and bmf transcription was increased, and both Bim and Bad were activated posttranslationally. Studies using RNAi and cells from gene-targeted mice revealed that Bim plays a major role in imatinib-induced apoptosis of Bcr/Abl+ leukemic cells and that the combined loss of Bim and Bad abrogates this killing. Loss of Bmf or Puma had no effect. Resistance to imatinib caused by Bcl-2 overexpression or loss of Bim (plus Bad) could be overcome by cotreatment with the BH3 mimetic ABT-737. These results demonstrate that Bim and Bad account for most, perhaps all, imatinib-induced killing of Bcr/Abl+ leukemic cells and suggest previously undescribed drug combination strategies for cancer therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-Associated Death Protein; Benzamides; Biphenyl Compounds; Cell Line, Tumor; Cell Transformation, Neoplastic; Cytotoxicity, Immunologic; Drug Resistance, Neoplasm; Fetus; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Liver; Membrane Proteins; Mice; Myeloid Progenitor Cells; Nitrophenols; Piperazines; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Sulfonamides