Ph-positive Leukemia Research Articles

Effect of D-mannose on Philadelphia chromosome-positive leukemia cells.

Although Abelson (ABL) tyrosine kinase inhibitors (TKIs) have demonstrated potency against chronic myeloid leukemia (CML), resistance to ABL TKIs can develop in CML patients after discontinuation of therapy. Glucose metabolism may be altered in CML cells because glucose is a key metabolite used by tumor cells. We investigated whether D-mannose treatment induced metabolic changes in CML cells and reduced CML growth in the presence of ABL TKIs. We investigated whether D-mannose treatment induced metabolic changes in CML cells and reduced CML growth in the presence of ABL TKIs. Treatment with D-mannose for 72h inhibited the growth of K562 cells. Combined treatment using ABL TKIs and D-mannose induced a significantly higher level of cytotoxicity in Philadelphia chromosome (Ph)-positive leukemia cells than in control cells. In the mouse model, severe toxicity was observed as evidenced by body weight loss in the ponatinib and D-mannose combination treatment groups. Our results indicate that metabolic reprogramming may be a useful strategy against Ph-positive leukemia cells. However, caution should be exercised during clinical applications.

Hypocellular Philadelphia chromosome‑positive mixed‑phenotype acute leukemia successfully treated with dasatinib: A case report

Both hypocellular leukemia and Philadelphia (Ph) chromosome-positive mixed-phenotype acute leukemia (MPAL) are rare subtypes of leukemia showing unfavorable outcomes and lacking established optimal management. Ph-positive leukemia most often presents with hypercellularity and hypoplasia is a rare condition. The present study reports an extremely rare case of hypocellular biclonal Ph-positive MPAL, which was diagnosed by biopsy and genetic analysis of bone marrow, and successfully treated with dasatinib and steroids. Briefly, a 77-year-old man presented with pancytopenia and flow cytometry of bone marrow could not be evaluated due to hypocellularity. The patient was finally diagnosed with hypocellular Ph-positive MPAL by genetic analysis and immunostaining of bone marrow biopsy. Although blood cells recovered with methylprednisolone pulse administration alone for concurrent optic neuritis, hematopoietic function rapidly normalized with dasatinib administered after definitive diagnosis of Ph-positive leukemia. Dasatinib and oral prednisolone were continued following methylprednisolone pulse administration and the patient achieved molecular complete remission (CR) on day 140 of treatment; molecular CR was maintained thereafter without any severe adverse events. In conclusion, the combination of dasatinib and a steroid may be one of the tolerable treatment options for elderly patients with hypocellular biclonal Ph-positive MPAL. Furthermore, genetic analysis and immunostaining of bone marrow biopsy can help with the diagnosis of leukemia with hypocellular bone marrow.

Rare Case of Pericentric Inversion, Inv(9)(p13q34) of the Der(9)t(9;22)(q34;q11) in A Patient with Chronic Myeloid Leukemia – A Case Report

As we know, the Philadelphia chromosome (Ph) is a highly specific marker for chronic myeloid leukemia (CML). This hematological disease is characterised by the formation of the BCR/ABL1 fusion gene, usually with typical translocation pattern including 9q34 and 22q11. In this paper we describe a 55 years old female patient with typical clinical and haematological signs of CML and a chromosome 9 differing from that which normally participates in translocation t(9;22). The karyotype of this Ph positive patient is characterised by pericentric inv(9)(p13q34) of the der(9)t(9;22)(q34;q11). Reverse transcriptase-polymerase chain reaction revealed a e14a2 type of BCR/ABL1 fusion transcript. As a consequence of this unusual translocation, FISH also found the separation of the ABL1/BCR1 fusion gene on chromosome 9. On reviewing the literature, to date only 10 Ph-positive leukemia patients have been noticed to have pericentric inversion inv(9)(p22q34)der(9)t(9;22)(q34;q11). No one case has been described with pericentric inversion inv(9)(p13q34) of the der(9)t(9;22)(q34;q11). This indicate that pericentric inv(9)(p13q34) of the der(9)t(9;22)(q34;q11) is a novel, rare, chromosomal abnormality in Ph-positive CML.

Aurora A Kinase As a Promising Therapeutic Target in Philadelphia Chromosome Positive Leukemia Cells

Introduction: ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, such as nilotinib and dasatinib have demonstrated the potency against chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (Ph+ALL) patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). Residual CML cells are present in bone marrow microenvironment. These cells are contained within a niche in the bone marrow and are often impervious to current treatments. Aurora kinases are novel family of serine/threonine kinases that play a critical role in regulating many of the processes that are pivotal to mitosis. Aurora kinases comprises three members, designated aurora A, B, and C. Amplification of aurora kinase gene is oncogenic and has been observed in several cancers including hematologic malignancies. Therefore, new approach to the treatment for Ph-positive cells with aurora kinase inhibitors may improve the outcome of leukemia patients.Materials and methods: In this study, we investigated whether aurora kinase inhibitor could suppress Ph-positive leukemia cells including T315I mutation and primary samples.Results: One of aurora kinase A inhibitor, alisertib inhibits proliferation of leukemia cells. We found alisertib treatment inhibited the growth of Ph-positive leukemia cells in a dose dependent manner and phosphorylation of aurora kinase A was reduced. Alisertib inhibits proliferation in imatinib resistant cells including T315I mutation. In the cell cycle analysis, alisertib induces a G2/M phase arrest in Ph-positive cells. Intracellular ROS levels were also increased in a dose dependent manner. Combined treatment of Ph-positive cells with ABL tyrosine kinase inhibitors, imatinib or ponatinib and alisertib caused significantly more cytotoxicity than each drug alone. Apoptotic and Caspase 3/7 positive cells were also increased. Ph-positive cells were treated with alisertib and cellular senescence was determined by senescence- associated β-galactosidase (SA-β-gal) staining. Senescent cells were increased in a time and dose dependent manner. SA-β-gal positive cells were reduced by N-Acetyl-L-cysteine treatment. Small interfering RNA mediated knockdown of the Aurora A gene causes a proliferation defect and enhances ABL TKI activity in Ph-positive cells. We next examined tumor formation in mice model. We injected subcutaneously 1×107 T315I mutant cells in nude mice. A dose of 20 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of alisertib inhibited tumor growth and reduced tumor volume compared with control mice. We also found that co-treatment with ponatinib and alisertib increased mouse survival. Co-treatment with ponatinib and alisertib showed reduced mouse spleen weight and size. The treatments were well tolerated with no animal health concerns observed.Conclusion: The results of our study indicate that the aurora kinase inhibitor may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. DisclosuresTauchi:Otsuka pharmaceutical KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau. Ohyashiki:Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Kyowa Hakko Kirin KK: Honoraria; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Fujirebio KK: Honoraria; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Taiho pharmaceutical KK: Honoraria; Alexion: Honoraria; MSD KK: Honoraria; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Fujirebio KK: Honoraria; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Chugai pharmaceutical KK: Research Funding; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Taiho pharmaceutical KK: Honoraria; Alexion: Honoraria; MSD KK: Honoraria; Kyowa Hakko Kirin KK: Honoraria; Chugai pharmaceutical KK: Research Funding.

The Pivotal Role of Nicotinamide Phosphoribosyl Transferase in Chronic Myeloid Leukemia Cells Under Hypoxic Condition

Introduction: ABL tyrosine kinase inhibitors (TKIs) improved outcomes for patients with chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive leukemia. However, ABL TKIs do not eliminate the leukemia stem cells (LSCs) in the bone marrow, which may represent the most important event in leukemia relapse after TKI discontinuation. Bone marrow is also considered a tissue with limited oxygen supply. Therefore, new approach against leukemia stem cells, which is presented in bone marrow under hypoxia, may improve the outcome of CML patients. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate limiting enzyme of the primary pathway for maintaining cellular NAD+ and that regulates intracellular adenosine triphosphate (ATP) levels in mammalian cells. It has been reported that NAMPT expression is upregulated in several human cancers. Materials and methods: In this study, we established ABL TKI resistantin vitrocell line models (K562 imatinib-R, K562 nilotinib-R, K562 ponatinib-R and Ba/F3 T315I) and used Ph-positive leukemia cell lines. We also investigated whether NAMPT inhibitor could suppress Ph-positive leukemia cells including T315I mutation and ABL TKI resistant under hypoxic condition. Results: In chemical library screen of compounds, NAMPT inhibitor, CHS828 is selected the candidate drug for ABL TKI resistant cells under hypoxic condition. Drug repositioning and repurposing can be an important part of any drug discovery. Therefore, we examined NAMPT efficacy by using Ph-positive leukemia cell lines. We first investigated the NAMPT expression by microarray gene expression data from the online Gene Expression Omnibus (GEO). Gene expression of NAMPT is increased in CML patients compared to normal samples from the public microarray datasets of GSE13159. Protein expression of NAMPT is found in Ph-positive leukemia cell lines including ABL TKI resistant cells. In hypoxia, gene expression of NAMPT was increased in Ph-positive cells compare to normoxic condition. We next examined efficacy of NAMPT inhibitor, CHS828 in Ph-positive leukemia cell lines. We found that CHS828 treatment for 72 h decreased cell viability of Ph-positive cell lines in a dose dependent manner. We also found that CHS828 inhibited the proliferation of ABL TKI resistant cells (K562 imatinib-R, K562 nilotinib-R, K562 ponatinib-R) and T315I mutant Ba/F3 cells under hypoxic condition. The rate of cell cycle progression was delayed and cells were blocked in G2/M phase. Nuclear factor-kappaB (NF-kB) is also implicated in cancer development. CHS828 inhibited constitutive NF-kB activity in the time and dose dependent manner. Intracellular ATP, which is the most important source of energy for cellular reactions, was drastically decreased after CHS828 treatment. NAD+ plays a vital role in diverse cellular processes that govern human health and disease. Intracellular NAD+ was decreased after CHS828 treatment. The wound healing effect was evaluated at 0, 24, 48, and 72 h by using NIH3T3 cells. In wound healing results, CHS828 treatment inhibited cellular migration of NIH3T3 cells. We next investigated the efficacy of ABL TKI and CHS828 against Ph-positive leukemia cell lines. ABL TKI and CHS828 treatment induced cellular growth inhibition compared with each drug alone. Caspase 3/7 activity was also increased after ABL TKIs and CHS828 treatment. ABL TKI and CHS828 reduced the colony formation ability of Ph-positive cells under hypoxic condition. Thein vivoefficacies of one of ABL TKI, ponatinib and CHS828 were evaluated in a mouse xenograft model. Ponatinib and CHS828 inhibited the growth of T315I mutant Ba/F3 cellsin vivomore than the control vehicle. We also found that co-treatment with ponatinib and CHS828 increased mouse survival. Combination treatment with ponatinib and CHS828 was also well tolerated, with no animal deaths in the treated mice. Conclusion: The results of our study indicate that the ABL TKI and CHS828 may be a powerful strategy against Ph-positive cells including ABL TKI resistant cells and provide the promising clinical relevance as a candidate drug for treatment of CML stem cells of the bone marrow microenvironment under hypoxic condition. Disclosures No relevant conflicts of interest to declare.

Molecular response and prognosis of pediatric patients with Ph-positive acute lymphoblastic leukemia treated by tyrosine kinase inhibitors with chemotherapy

Objective To explore the molecular response and prognostic factors of pediatric patients with Ph-positive acute lymphoblastic leukemia (Ph+ ALL) treated by tyrosine kinase inhibitors (TKI) with chemotherapy in TKI era. Methods The clinical data of children newly diagnosed with Ph+ ALL admitted at Department of Pediatrics, Peking University People′s Hospital from August 2006 to February 2017 were retrospectively reviewed.The molecular biological characteristics and survival prognosis of the 30 patients who received continuous TKI with chemotherapy from early induction combined and no subsequent transplantation were analyzed. Results The 30 patients with Ph+ ALL had 19 males and 11 females with a median age of 8-year-old (ranging from 2 to 16 years). The complete remission (CR) rate after the first cycle of induction chemotherapy was 96.7% (29/30 cases), with overall CR rate of 100.0%; Before treatment, the mean level of BCR/ABL mRNA in the 30 patients was 73.2% (0.12%-160.60%) and the level declined significantly with the progression of chemotherapy courses, reaching the plateau stage at the 6th month of chemotherapy (Z=-1.922, P>0.05); nine patients had recurrence, with a median recurrence time of 7 months (3.7-58.8 months). Univariate analysis showed that age (P<0.05), the lever of minimal residual disease (MRD) after induction chemotherapy (P<0.01) and the MRD level at the 3th month of induction chemotherapy (P<0.01) affected the recurrence rate.The median follow-up time of 30 patients was 42.6 months (6.4-96.5 months), and the 3-year overall survival (OS) rate and event-free survival (EFS) rate were (78.6±7.8)% and (72.4±8.4)%, respectively; Cox multivariate analysis showed that the initial white blood cell count ≥34.0×109/L (OR=11.955, 95% CI: 1.075-132.899, P<0.05) and BCR/ABL mRNA reduction less than 3 log from baseline [major molecular response (MMR)] at the 3th month of induction chemotherapy (OR=8.563, 95% CI: 1.254-58.478, P<0.05) were independent risk factors affecting the 3-year EFS rate.In addition, the initial white blood cell count ≥34.0×109/L (OR=14.327, 95% CI: 1.843-243.592, P<0.05) was also an independent risk factor affecting the 3-year OS rate. Conclusions The application of TKI can significantly deepen the molecular response of Ph+ ALL in children.In the TKI era, the initial white blood cell count ≥ 34.0×109/L and BCR/ABL mRNA reduction less than 3 log from baseline (MMR) at the 3th month of induction chemotherapy are independent risk factors for the long-term survival of pediatric Ph+ ALL. Key words: Philadelphia chromosome; Acute lymphoblastic leukemia; Child; Tyrosine kinase inhibitor; Mole-cular response

Deubiquitylase USP25 prevents degradation of BCR-ABL protein and ensures proliferation of Ph-positive leukemia cells.

Fusion genes resulting from chromosomal rearrangements are frequently found in a variety of cancer cells. Some of these are known to be driver oncogenes, such as BCR-ABL in chronic myelogenous leukemia (CML). The products of such fusion genes are abnormal proteins that are ordinarily degraded in cells by a mechanism known as protein quality control. This suggests that the degradation of BCR-ABL protein is suppressed in CML cells to ensure their proliferative activity. Here, we show that ubiquitin-specific protease 25 (USP25) suppresses the degradation of BCR-ABL protein in cells harboring Philadelphia chromosome (Ph). USP25 was found proximal to BCR-ABL protein in cells. Depletion of USP25 using shRNA-mediated gene silencing increased the ubiquitylated BCR-ABL, and reduced the level of BCR-ABL protein. Accordingly, BCR-ABL-mediated signaling and cell proliferation were suppressed in BCR-ABL-positive leukemia cells by the depletion of USP25. We further found that pharmacological inhibition of USP25 induced rapid degradation of BCR-ABL protein in Ph-positive leukemia cells, regardless of their sensitivity to tyrosine kinase inhibitors. These results indicate that USP25 is a novel target for inducing the degradation of oncogenic BCR-ABL protein in Ph-positive leukemia cells. This could be an effective approach to overcome resistance to kinase inhibitors.

Nilotinib treatment induced large granular lymphocyte expansion and maintenance of longitudinal remission in a Philadelphia chromosome-positive acute lymphoblastic leukemia

It is well known that the second-generation tyrosine kinase inhibitor dasatinib evokes an immunological reaction as an off-target effect and induces large granular lymphocytes (LGLs) expansion in 30% of patients. However, LGLs expansion in nilotinib-treated patients is rare. We report the case of a 65-year-old patient with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) who showed LGLs expansion during nilotinib treatment. The patient achieved complete remission (CR) after multi-agent chemotherapy combined with dasatinib treatment. However, ALL relapsed in the central nervous system and bone marrow when treatment was interrupted due to interstitial pneumonia. Nilotinib treatment was subsequently started and the patient achieved second CR. Marked peripheral blood lymphocytosis emerged after the start of nilotinib treatment. CD8 + CD57 + cytotoxic T cells were predominantly expanded and showed strong cytocidal activity against K562 Ph-positive leukemia cells. These results suggest that similar to dasatinib, nilotinib can induce LGLs expansion, possibly contributing to long-term remission in patients with Ph-ALL.

FLT3-ITD Activates RSK1 to Enhance Proliferation and Survival of AML Cells by Activating mTORC1 and eIF4B Cooperatively with PIM or PI3K and by Inhibiting Bad and BIM.

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously found that FLT3-ITD activates the mTORC1/S6K/4EBP1 pathway cooperatively through the STAT5/PIM and PI3K/AKT pathways to promote proliferation and survival by enhancing the eIF4F complex formation required for cap-dependent translation. Here, we show that, in contrast to BCR/ABL causing Ph-positive leukemias, FLT3-ITD distinctively activates the serine/threonine kinases RSK1/2 through activation of the MEK/ERK pathway and PDK1 to transduce signals required for FLT3-ITD-dependent, but not BCR/ABL-dependent, proliferation and survival of various cells, including MV4-11. Activation of the MEK/ERK pathway by FLT3-ITD and its negative feedback regulation by RSK were mediated by Gab2/SHP2 interaction. RSK1 phosphorylated S6RP on S235/S236, TSC2 on S1798, and eIF4B on S422 and, in cooperation with PIM, on S406, thus activating the mTORC1/S6K/4EBP1 pathway and eIF4B cooperatively with PIM. RSK1 also phosphorylated Bad on S75 and downregulated BIM-EL in cooperation with ERK. Furthermore, inhibition of RSK1 increased sensitivities to BH3 mimetics inhibiting Mcl-1 or Bcl-2 and induced activation of Bax, leading to apoptosis, as well as inhibition of proliferation synergistically with inhibition of PIM or PI3K. Thus, RSK1 represents a promising target, particularly in combination with PIM or PI3K, as well as anti-apoptotic Bcl-2 family members, for novel therapeutic strategies against therapy-resistant FLT3-ITD-positive AML.

PARP Inhibition Sensitize BCR-ABL1 Positive Cel

Introduction: BCR-ABL1 play a key role in the development of chronic myelogenous leukemia and a part of Ph1 positive acute lymphoblastic leukemia (ALL). BCR-ABL1 functions as a tyrosine kinase. Whereas, BCR-ABL1 induces genomic instability by downregulation of BRCA1. An innate error of BRCA1, a molecule involved in the homologous recombination repair pathway, causes hereditary breast and ovarian cancer. PARP inhibitor (PARPi) induces synthetic lethality in BRCA defective cell. Therefore, PARP inhibitor is expected to induce efficient cell death with BCR-ABL1 positive cell. In addition, in some previous reports, reduction of PARP1 activity leads to the upregulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and BCR-ABL1 tyrosine kinase activates PI3K/AKT pathway. These findings suggest activation of the PI3K/AKT pathway leading to PARPi resistance in BCR-ABL1 positive leukemic cells. Here, we demonstrate that PARP inhibition attenuates BCR-ABL1 mediated leukemogenesis and aberration of factors associated with PARP inhibitor resistance induces cell death to fully transformed leukemic cells. Method: Bone marrow-derived mononuclear cells (MNC) from wild type mice and BCR-ABL1 transgenic (Tg) mice were exposed to PARPi in vivo, and cell death was analyzed Annexin-V positivity. PARPi sensitivity to BCR-ABL1 expressed cell was also investigated in vivo bone marrow transplantation model using mouse hematopoietic stem cell (HCS) infected with BCR-ABL1 expressing retrovirus. To evaluate more precisely the results obtained in vitro and in vivo transplantation model, the genetical approach was also performed. The Parp1 knockout (KO) mice were crossed with BCR-ABL1 Tg mice. Then, Leukemia development and subsequent mouse death were observed. In vitro, HR activity was examined using DR-GFP assay. Genomic instability was investigated using the breakage-fusion-bridge (BFB) generation.Maintenance of HSC as a progenitor of the leukemic cell was analyzed by repopulation activity using colony assay. The growth-inhibitory effect was assessed using BCR-ABL positive cell lines with PARPi and PI3K inhibitor. Results: BCR-ABL1 Tg mice derived MNC showed more hypersensitivity to PARPi. Mouse HCS was infected with BCR-ABL1 expressing retrovirus and transplanted lethally Olaparib or vehicle was administrated intraperitoneal injection one day after transplantation. BCR-ABL1 mediated leukemic death was observed 1 month after transplantation in sham-treated mouse, whereas, Olaparib treated mouse did not develop BCR-ABL1 mediated leukemia. Parp1 KO BCR-ABL1 Tg mice attenuated leukemia development and extended their survival compared with BCR-ABL1 Tg mice. In vitro experiment revealed HR activity was down-regulated by BCR-ABL1 expression in DR-GFP assay. The number of BFB generation was increased in BCR-ABL1 Tg with Parp1 KO background. The colony-forming activity of BCR-ABL1 positive HSC was totally abolished by PARP inhibition after 3 times serial replating, whereas sham-treated HSC retained repopulation activity. However, the effect of PARPi on BCR-ABL positive leukemic cell lines was controversial. Therefore, leukemic cell lines were treated with the PARPi and inhibitors toward the molecules associated with PARPi resistance. As a result, a combination of PARPi with PI3K inhibitor effectively induce cell death in PARPi resistant BCR-ABL1 positive leukemic cell lines. Conclusion and discussion: Tyrosine kinase inhibitor (TKI) is the gold standard of the therapeutic option of BCR-ABL1 positive leukemia. However, TKI monotherapy is not sufficient for complete eradication of leukemic cells. It is highly expected that molecules effectively induce cell death to leukemic cells combined with TKI. PARPi would be one of these candidates. However, PARPi could not induces efficient death in all of the cancer cells that carry the mutation of molecules associated with the HR defect. Comprehensive genetic analysis to reveal PARPi resistance is important for HRR defective cancer cells. Combination therapy of PARPi and inhibitorstoward the molecules associated with PARPi resistance would be a good therapeutic option for Ph1 positive leukemia. Disclosures No relevant conflicts of interest to declare.

The Role of Hypoxia on ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells

Introduction: ABL tyrosine kinase inhibitors (TKIs) improved outcomes for patients with chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive leukemia, however, some patients are still resistance to ABL TKIs. One of the most common mechanisms involves point mutations in the kinase domain of BCR-ABL1, however, mechanisms of intrinsic resistance without point mutation of ABL kinase domain are not fully understood. Moreover, ABL TKIs cannot cure the Ph-positive leukemia patients because of leukemia stem cells in the bone marrow niche. Therefore, new approach against leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Hypoxia is an important component of the bone marrow microenvironment. Because oxygen tension plays a key role in driving normal hematopoiesis, leukemia stem cells may be maintained in hypoxic areas of the bone marrow. Materials and methods: In this study, we established ABL TKI-resistant in vitro cell line models (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R and Ba/F3 T315I). We investigated gene expression profiles in cultured ABL TKI resistant cells and parental cell line, K562 in normoxia and hypoxia condition by DNA microarray. Results: We first investigated gene expression profiles in cultured K562 cells in hypoxia condition. We found gene expression of insulin-like growth factor 1 (IGF1) was increased K562 cells in hypoxia condition by DNA microarray. We next examined ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R) in this study. We could not detect the BCR-ABL point mutation in ABL TKI resistant cells. We found gene expression of insulin-like growth factor 1 (IGF1) receptor (IGF1R) was increased ABL TKI resistant K562 cells. IGF1R gene amplification was confirmed by RT-PCR analysis. IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). One of IGFBP, IGFBP5 is related to imatinib sensitivity and resistant in chronic myeloid leukemia (CML) patients (GSE12211). In hypoxia condition, several IGFBPs were also increased in ABL TKI resistant cells. IGF cause intracellular signaling that ultimately results in cellular growth and proliferation. Thus, we initially examined whether addition of IGF1R inhibition could enhance ABL TKIs sensitivity. One of IGF1R inhibitor, linsitinib was inhibited ABL TKI resistant cells and parental cell line, K562 in hypoxia condition. ABL TKI resistant cell lines were more sensitive against linsitinib. Combined treatment of ABL TKI resistant cells and K562 cells with ABL TKIs and linsitinib caused more cytotoxicity than each drug alone in hypoxia condition. Caspase 3/7 activity and cellular cytotoxicity was also increased after ABL TKIs and linsitnib treatment. In the colony formation method, the number of cell colonies were also reduced in hypoxia condition. Intracellular ATP levels have been implicated in vitro as a determinant of cell death by apoptosis. The concentrations of intracellular ATP were reduced after ABL TKIs and linsitinib. We next blocked IGF1R function by small interfering RNA (siRNA). SiRNA transfected cells were reduced cellular proliferation. We also found drug sensitivity of the cells to the imatinib was increased compared to mock-transfected cells. Apoptotic cells and caspase 3/7 activity were increased after imatinib treatment in siRNA transfected cells. Conclusion: The IGF1 pathway is involved in Ph-positive leukemia cells in hypoxia condition and ABL TKI resistant in CML cells. We also provide the promising clinical relevance as a candidate drug for treatment of residual leukemia cells in bone marrow niche which is in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

Quinacrine Depletes BCR-ABL and Suppresses Ph-Positive Leukemia Cells

Drug resistance to TKIs and the existance of CML leukemia stem cells is an urgent problem. In this study, we demonstrate that quinacrine (QC) induces apoptosis in BCR-ABL positive CML and acute lymphoblastic leukemia (ALL) cells. Interestingly, QC inhibits the colony formation of primary CD34+ progenitor/stem leukemia cells from CML patients. QC targets RNA polymerase I, which produces ribosomal (r)RNA, involving in protein translation process. Also, QC treatment prolongs CML-like mice survival and inhibits K562 tumor growth in vivo. In conclusion, we demonstrate that QC depletes BCR-ABL protein and suppresses Ph-positive leukemia cells in vitro and in vivo.

PF400 ANTI-LEUKEMIA EFFECT OF DISULFIRAM AND ASCORBATE IN ABL TYROSINE KINASE INHIBITOR RESISTANT LEUKEMIA CELLS

Background: ABL tyrosine kinase inhibitors (TKIs) such as imatinib improved outcomes for patients with Philadelphia chromosome (Ph)-positive leukemia patients, however, some patients are still resistance to ABL TKIs. One of the most common mechanisms involves point mutations in the kinase domain of BCR-ABL1, however, mechanisms of intrinsic resistance without point mutation of ABL kinase domain are not fully understood. Therefore, new approach against ABL TKI resistant cells may improve the outcome of Ph-positive leukemia patients. Aims: Because disulfiram (DSF), which is used to treat alcohol dependence in clinically, has been reported to have anti-tumor effects in various cancers, DSF may be the promising therapeutic value in ABL TKI resistant leukemia cells. Methods: In this study, we established ABL TKI resistant in vitro cell line models. We also investigated whether DSF could suppress Ph-positive leukemia cells including T315I mutation and ABL TKI resistant cells. Results: Drug repositioning and repurposing can be an important part of any drug discovery. In the drug screening system, the alcohol-aversion drug disulfiram, which is already approved for the treatment of alcohol dependence, is selected the candidate drug for ABL TKI resistant cells. Therefore, we examined DSF efficacy by using Ph-positive leukemia cells. We found that DSF treatment for 72 h decreased cell viability in a dose-dependent manner. We also found that DSF inhibited the proliferation of ABL TKI resistant cells (K562 ponatinib-R, K562 nilotinib-R) and T315I mutant Ba/F3 cells. Vitamin C (ascorbate) has also been well documented to reduce the incidence of malignancies in humans. We next investigated the efficacy of DSF and ascorbate against ABL TKI resistant and T315I mutant Ba/F3 cells. DSF and ascorbate treatment induced cellular growth inhibition compared with each drug alone. Most strikingly, cell death started in ABL TKI resistant cell lines within 3 to 6 h of treatment, indicating a first response to DSF and ascorbate lesions, to cell fate decision. Apoptotic cells and cellular cytotoxicity was also increased after DSF and ascorbate treatment. In the Colony Formation method, the number of cell colonies are also reduced. We next investigated the signaling pathway. Nuclear factor-kappaB (NF-kB) is also implicated in cancer development. DSF and ascorbate inhibited constitutive NF-kB activity. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased after DSF and ascorbate treatment. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential (Δψm) in cell death induced by DSF and ascorbate on T315I mutant Ba/F3 cells. The in vivo efficacies of DSF and ascorbate were evaluated in a mouse xenograft model. DSF and ascorbate inhibited the growth of T315I mutant Ba/F3 cells in vivo more than the control vehicle did. We also found that co-treatment with DSF and ascorbate increased mouse survival. Combination treatment with DSF and ascorbate was also well tolerated and no animal death in treated mice. Summary/Conclusion: The results of our study indicate that the DSF and ascorbate may be a powerful strategy against ABL TKI resistant cells including T315I mutation and provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients.

Interleukin 1 Beta As a Promising Therapeutic Target in ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells

Introduction: Although ABL tyrosine kinase inhibitor (TKI), imatinib, nilotinib and dasatinib have demonstrated the potency against chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (Ph+ALL) patients, resistance to ABL TKI can develop in the many patients. It has already reported that ABL kinase domain mutations have been implicated in the pathogenesis of ABL TKI resistance, however, it is fully not known the molecular mechanism of drug resistance ABL TKIs. Therefore, new approach against ABL TKI resistant cells may improve the outcome of Ph-positive leukemia patients. Interleukin-1 (IL-1) is a proinflammatory cytokine and central mediator of innate immunity. IL-1β also controls essential cell responses. Because enhanced IL-1β signaling is reported in patients of hematological malignancies, IL-1β may be the promising therapeutic value in ABL TKI resistant CML patients.Materials and methods: In this study, we established ABL TKI-resistant in vitro cell line models (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R). We also investigated whether IL-1β was involved in ABL TKI resistant Ph-positive leukemia cells and cytokines were induced by IL-1β in human umbilical vein endothelial cells (HUVEC).Results: We analyzed the relationship of IL-1β signaling pathways and ABL TKI sensitivity by microarray gene expression data from the online Gene Expression Omnibus (GEO). IL-1β is related to imatinib sensitivity and resistant in CML patients from the public microarray datasets of GSE14671. We next examined ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R) in this study. BCR-ABL point mutation was not found in ABL TKI resistant cells. BCR-ABL expression levels were not increased in ABL TKI resistant K562 cells. These cells were highly resistant to ABL TKIs compare to K562 cells (K562 imatinib-R: imatinib 2μM, nilotinib-R: nilotinib 2μM, dasatinib-R: dasatinib 100nM, ponatinib-R: ponatinib 50nM). We investigated gene expression profiles in cultured ABL TKI resistant K562 cells by DNA microarray. We found gene expression of IL-1β and IL-1β mediated signaling pathway was increased ABL TKI resistant K562 cells. IL-1β gene amplification was confirmed by RT-PCR analysis. Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) acts as a central mediator of inflammatory responses. Because bortezomib is a proteasome inhibitor whose anti-cancer action is partly mediated through inhibition of NF-κB, we examined the bortezomib in ABL TKI resistant cells. Combined treatment of ABL TKI resistant cells with ponatinib or imatinib and bortezomib caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. ABL TKIs are also associated with vascular adverse events (VAEs) in CML, we next investigated the in vitro effects of ABL TKIs on cultured HUVEC. We found gene expression of IL-1β was increased after ABL TKI especially dasatinib and ponatinib treatment. IL-1β was increased in the cell culture supernatant after ABL TKIs treatment. In the immunoblot analysis, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation was increased by IL-1β in the time dependent manner. We also found that gene expression of IL-6, IL-8, intercellular adhesion molecule 1 (ICAM1) and monocyte chemotactic protein-1 (MCP-1) was enhanced by IL-1β stimulation.Conclusion: The IL-1β signaling pathway is involved in ABL TKI sensitivity and drug resistant in CML cells and plays a key role in cytokine production of the HUVEC. We also provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients. DisclosuresOhyashiki:Asahikase: Research Funding; Taiho Pharmaceutical KK: Honoraria, Research Funding; Dainippon Sumitomo KK,: Honoraria, Research Funding; MSD,: Honoraria, Research Funding; Bristol Meyer Squibb KK,: Honoraria, Research Funding; Ono Pharmaceutical KK,: Honoraria, Research Funding; Celegene KK,: Honoraria, Research Funding; Pfizer KK,: Honoraria, Research Funding; Kyowakko Kirin KK,: Research Funding; Nihon-Seiyaku,: Research Funding; Eizai,: Research Funding; Chugai KK,: Honoraria, Research Funding; Takeda Pharmaceutical KK,: Honoraria, Research Funding; Asteras KK,: Research Funding; Jansen Pharma KK,: Research Funding; Nippon-shinyaku,: Honoraria, Research Funding; Novartis KK,: Honoraria, Research Funding.

The Type of BCR/ABL1 Fusion Transcript Does Not Affect Therapy Effectiveness of Ph-Positive Acute Lymphocytic Leukemia in the Era of Tyrosine Kinase Inhibitors in Russian Trial Ph+ALL

The Type of BCR/ABL1 Fusion Transcript Does Not Affect Therapy Effectiveness of Ph-Positive Acute Lymphocytic Leukemia in the Era of Tyrosine Kinase Inhibitors in Russian Trial Ph+ALL

Transcriptional activation of the miR-17-92 cluster is involved in the growth-promoting effects of MYB in human Ph-positive leukemia cells.

MicroRNAs, non-coding regulators of gene expression, are likely to function as important downstream effectors of many transcription factors including MYB. Optimal levels of MYB are required for transformation/maintenance of BCR-ABL-expressing cells. We investigated whether MYB silencing modulates microRNA expression in Philadelphia-positive (Ph+) leukemia cells and if MYB-regulated microRNAs are important for the “MYB addiction” of these cells. Thirty-five microRNAs were modulated by MYB silencing in lymphoid and erythromyeloid chronic myeloid leukemia-blast crisis BV173 and K562 cells; 15 of these were concordantly modulated in both lines. We focused on the miR-17-92 cluster because of its oncogenic role in tumors and found that: i) it is a direct MYB target; ii) it partially rescued the impaired proliferation and enhanced apoptosis of MYB-silenced BV173 cells. Moreover, we identified FRZB, a Wnt/β-catenin pathway inhibitor, as a novel target of the miR-17-92 cluster. High expression of MYB in blast cells from 2 Ph+leukemia patients correlated positively with the miR-17-92 cluster and inversely with FRZB. This expression pattern was also observed in a microarray dataset of 122 Ph+acute lymphoblastic leukemias. In vivo experiments in NOD scid gamma mice injected with BV173 cells confirmed that FRZB functions as a Wnt/β-catenin inhibitor even as they failed to demonstrate that this pathway is important for BV173-dependent leukemogenesis. These studies illustrate the global effects of MYB expression on the microRNAs profile of Ph+cells and supports the concept that the “MYB addiction” of these cells is, in part, caused by modulation of microRNA-regulated pathways affecting cell proliferation and survival.

Peroxisome Proliferator-Activated Receptors: Targets for the Treatment of Philadelphia Chromosome-Positive Leukemia Cells

Abstract Introduction: The efficacy of chemotherapy in patients with Philadelphia chromosome (Ph)-positive leukemia has been dramatically improved by the development of small-molecule ABL tyrosine kinase inhibitors (TKIs), such as imatinib. However, resistance to ABL-TKI can develop in patients with chronic myeloid leukemia (CML) because of the presence of leukemia stem cells (LSCs). Thus, a new approach against LSCs may improve the outcome of patients with Ph-positive leukemia. Peroxisome proliferator-activated receptors (PPARs) constitute a superfamily of nuclear hormone receptors and regulate the transcription of target genes. Three subtypes of PPARs (α, γ, and δ/β) have been identified. PPARγ is expressed in both adipose tissue and cancer cells. A PPARγ agonist, pioglitazone, is used in the treatment of diabetes patients. It has recently been reported that PPARγ agonists eradicate leukemia stem cells in patients with CML. We hypothesized that targeting PPARγ, in combination with ABL-TKIs, would result in enhanced therapeutic activity in Ph-positive leukemia cells. Materials and Methods: We investigated the effect of a PPARγ agonist, pioglitazone, on Ph-positive leukemia cell lines and primary samples. Results: We examined the expression of PPARs in Ph-positive leukemia cells and found that PPARα and PPARγ were expressed in Ph-positive leukemia cells, including primary samples. However, we could not detect PPARα and PPARγ expressions in normal CD34-positive cells by immunoblot analysis; this suggests that PPARα and PPARγ may be targets of Ph-positive leukemia cells. Next, we examined the efficacy of pioglitazone. Pioglitazone treatment for 72 h inhibited the growth of Ph-positive cells, including T315I-mutant cells. In contrast, pioglitazone did not inhibit normal CD34-positive cells. We then examined intracellular signaling. The phosphorylation of BCR-ABL and Crk-L was not reduced after pioglitazone treatment. In contrast, the phosphorylation of AMP-activated protein kinase (AMPK) was increased, whereas the phosphorylation of S6 ribosomal protein and signal transducer and activator of transcription 5 (STAT5) was reduced. The combined treatment of Ba/F3 T315I cells with ponatinib and 30 µM pioglitazone resulted in greater cytotoxicity. Phosphorylation of S6 ribosomal protein and STAT5 was reduced, whereas AMPK phosphorylation was increased. Cleaved poly (ADP-ribose) polymerase (PARP) was increased after treatment with ponatinib and pioglitazone. Thereafter, we investigated anti-angiogenic effects on human umbilical vein endothelial cells (HUVECs) because expression of PPARα and PPARγ was reported in HUVECs in previous studies. Pioglitazone inhibited tube formation of HUVECs through vascular endothelial growth factor (VEGF) in the extracellular matrix gel, suggesting that pioglitazone inhibits angiogenesis in vitro . Further, we found that the treatment with ponatinib or imatinib and/or pioglitazone inhibited the growth of primary samples, including CD34-positive CML cells; however, these drugs did not affect the normal cells. The phosphorylation of STAT5 was reduced and AMPK phosphorylation was increased. We next conducted a case study. A 61-year-old patient was treated with nilotinib (800 mg/day), and BCR-ABL levels in the peripheral blood cell samples of the patient were monitored using real-time polymerase chain reaction (PCR); however, the international scale (IS) of BCR-ABL remained elevated. Oral pioglitazone (15 mg, once daily) was administered because the patient had high blood glucose levels for more than 1 year. Initiation of pioglitazone therapy resulted in reduction of IS% of BCR-ABL. Conclusion: Our study indicated that administration of the PPARγ agonist, pioglitazone, may enhance the effects of ABL-TKI and is effective in suppressing angiogenesis mediated by VEGF. Thus, PPARγ agonists may possess promising clinical relevance as a candidate therapeutic agent for Ph-positive leukemia in the future. Disclosures Tauchi: Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau. Ohyashiki: Phizer KK: Honoraria, Research Funding, Speakers Bureau; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Kyowa Hakko Kirin KK: Honoraria; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Honoraria; MSD KK: Honoraria; Fujirebio KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Chugai pharmaceutical KK: Research Funding; Taiho pharmaceutical KK: Honoraria; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Alexion: Honoraria; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau.

Characteristics and mutation analysis of Ph-positive leukemia patients with T315I mutation receiving tyrosine kinase inhibitors.

BackgroundTKIs are the first-line treatment for patients with Ph-positive (Ph+) leukemia. However, drug resistance is frequently observed, mainly due to mutations within the breakpoint cluster region-Abelson leukemia virus (BCR-ABL) kinase domain. The T315I substitution confers complete resistance to TKIs. The aim of this study was to analyze the clinical characteristics of 17 patients with T315I mutation after TKI treatment and provide a basis for prognosis.Patients and methodsThe clinical data of 17 TKI-resistant Ph+ leukemia patients who were found to have a ABL kinase domain mutation from September 2008 to January 2017 were collected. Karyotypes and BCR-ABL fusion gene were analyzed by R-banding and fluorescence in situ hybridization, respectively. Total RNA was extracted by TRIzol reagent, and the ABL kinase domain mutation was detected by direct sequencing.ResultsA total of 17 patients reached effective remission including major molecular response and complete cytogenetic response. However, all the patients subsequently developed a T315I mutation after treatment with TKIs. The rate of the BCR-ABL fusion gene in most of the patients who developed the T315I mutation was significantly higher than that before the mutation. At initial diagnosis, patients average platelet count was 149.7×109/L, whereas the average platelet count was only 53.88×109/L after the T315I mutation (P<0.01). The results also showed that the survival time of patients with a high proportion of blast cells or a high number of white blood cells was obviously shortened.ConclusionPatients platelet count decreased when detected with the T315I mutation compared with the initial diagnosis. Combined use of different TKIs and complex chromosomal karyotypes may promote the development of the T315I mutation. When the ratio of blast cells was >50% and the number of white blood cells was >20×109/L, poor survival prognosis was observed.

Pезультаты аллогенной трансплантации гемопоэ - тических стволовых клеток в смешанной когорте больных с Ph-позитивным острым лимфобластным лейкозом

Pезультаты аллогенной трансплантации гемопоэ - тических стволовых клеток в смешанной когорте больных с Ph-позитивным острым лимфобластным лейкозом

Anti-Leukemic Effects of Venetoclax on Philadelphia Chromosome Positive Leukemia Cells

Introduction: Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). Although ABL tyrosine kinase inhibitors (ABL TKI) such as imatinib, dasatinib and nilotinib have improved CML treatment, such therapies cannot cure patients with Philadelphia chromosome (Ph)-positive leukemia because of leukemia stem cells. Moreover, some patients develop BCR-ABL point mutations including T315I and become resistant to ABL TKI therapy. These leukemia stem cells are contained within a niche in the bone marrow and are often impervious to current treatments. Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. B cell lymphoma 2 (BCL-2) protein families are key regulator of apoptosis and highly promising targets for the development of anti-cancer treatment. Venetoclax, also known as ABT-199 is a selective, orally bioavailable BCL-2 inhibitor. Venetoclax is investigated in a pivotal phase 3 clinical trial against hematological malignancies such as chronic lymphocytic leukemia (CLL) and approved for the treatment of patients with CLL.Materials and methods: In this study, we investigated whether venetoclax could suppress Ph-positive leukemia cells including T315I mutation and primary samples.Results: BCL-2 expression was found in Ph-positive leukemia cells including primary samples, however, BCL-2 expression was reduced in K562 cells. We found 72 h venetoclax treatment inhibited the growth of Ba/F3 BCR-ABL and KCL-22 cells in a dose dependent manner. However, venetoclax activity was reduced in K562 cells. We examined the intracellular signaling after treatment of venetoclax. Phosphorylation of BCR-ABL and Crk-L was not reduced. However, activity of caspase 3, poly (ADP-ribose) polymerase (PARP) was increased. We next investigated the efficacy between ABL TKI and venetoclax by using these cell line. Combined treatment of Ba/F3 BCR-ABL cells with imatinib and venetoclax caused significantly more cytotoxicity than each drug alone. Apoptotic cells were also increased. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved caspase 3 and PARP activity was increased after imatinib and venetoclax treatment. We investigated the venetoclax activity against T315I positive cells. Venetoclax potently induced cell growth inhibition of Ba/F3 T315I mutant cells in a dose dependent manner. Combined treatment of Ba/F3 T315I mutant cells with ponatinib and venetoclax caused significantly more cytotoxicity than each drug alone. Apoptotic cells were also increased. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP was increased after ponatinib and venetoclax treatment. To assess the activity of ponatinib and venetoclax, we examined tumor formation in mice model. We injected subcutaneously 1×107 Ba/F3 T315I mutant cells in nude mice. A dose of 20 mg/kg/day p.o of ponatinib and 50 mg/kg/day p.o of venetoclax inhibited tumor growth and reduced tumor volume compared with control mice. In the immunohistochemical analysis, we found that tumors in mice treated with ponatinib and venetoclax exhibited an increase in apoptotic cells. We also found that co-treatment with ponatinib and venetoclax increased mouse survival. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of venetoclax exhibits cell growth inhibition against CD34 positive CML samples.Conclusion: The results of our study indicate that the BCL-2 inhibitor venetoclax may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. DisclosuresTauchi:Pfizer Inc.: Research Funding. Ohyashiki:Novartis International AG,: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding.