Treatment Of Ph Research Articles

Nilotinib Results in Improved Rates of Molecular Response in Turkish Newly Diagnosed CML-CP Patients: A 24-Month Update

IntroductionNilotinib, a more potent and selective BCR-ABL-1 inhibitor than imatinib, is approved by the FDA and EMA 1) for the treatment of chronic and accelerated phase Philadelphia chromosome positive (Ph+) Chronic Myeloid Leukemia (CML) patients resistant or intolerant to prior therapy including imatinib and 2) for the treatment of newly-diagnosed Ph+ CML patients in the chronic phase (CML-CP). The aim of the present study was to evaluate the efficacy and safety of nilotinib in a Turkish population of newly-diagnosed Ph+ CML-CP patients.MethodsThe study was a multicenter, open-label, single-arm phase II clinical trial. All patients were to be treated with nilotinib (AMN107, Tasigna®) 300 mg BID for 24 months.ResultsAs of April 30, 2014, of the 96 patients out of a total 112 enrolled that had 24 month follow-up, 77 completed active treatment period. General characteristics of the patients at baseline are presented in Table 1. [Display omitted] Data are expressed as median (minimum-maximum) or number (%), where appropriate.Molecular response by 24 months is presented in Table 2. [Display omitted] Data are presented as mean±standard deviation or number (%), where appropriate.MMR (Major molecular rate): BCR-ABL/control gene ratio of ≤%0.1 measured by RQ-PCR as %* The number of patients who were still on treatment at 3rd month.Cumulative MMR rates at various time points are shown in Figure 1. [Display omitted] A total of 486 adverse events (AEs) occurred; 93 AE led to discontinuation of the drug temporarily or permanently. Out of 96 patients 39 patients have temporally or permanently discontinued treatment. Permanent discontinuation rate due to adverse event was 9.6% (9 patients) which was the most common reason among all patients (19.8% - 19 patients) who permanently discontinued during 24 months. Thrombocytopenia was the most frequent (10.4%) AE, followed by hyperbilirubinemia (8.3%) and increased lipase level (7.3%) 19.8% of the patients entered in the trial had history of cardiovascular disease prior to trial entry and during the study cardiovascular events have been reported at 20.8% of patients. Despite published peripheral arterial occlusive disease (PAOD) reports related to nilotinib usage, there were no PAOD events reported at our cohort.ConclusionsAt 24 months, in Turkish patients with newly diagnosed CML, the cumulative MMR rate was 80.2%. Month 3 MMR rate was 86.5% and median time to MMR was 260.0±157.5 days. Out of 12 patients whose bcr-abl levels were above 10% at 3rd month, only 5 of them could reach to MMR by 24 months. Only one progression occurred, during the first year of therapy. These results suggest that high efficacy was achieved with nilotinib, an approved first-line therapy for newly diagnosed chronic phase of CML (CP-CML).The results of the present study revealed that efficacy and safety of nilotinib in Turkish cohort are similar to those reported in ENESTnd. This study contribute to well established nilotinib profile for chronic phase CML patients which is a licensed alternative for the treatment of newly diagnosed Ph+ CML-CP in Turkey. DisclosuresSaydam:Novartis Pharmaceuticals Corporation, Turkey: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity’s Board of Directors or advisory committees. Yavuz:Novartis Pharmaceuticals Corporation, Turkey: Membership on an entity’s Board of Directors or advisory committees. Ali:Novartis Pharmaceuticals Corporation, Turkey: Membership on an entity’s Board of Directors or advisory committees. Guvenc:Novartis Pharmaceuticals Corporation, Turkey: Membership on an entity’s Board of Directors or advisory committees. Sonmez:Novartis Pharmaceuticals Corporation, Turkey: Membership on an entity’s Board of Directors or advisory committees. Akkaynak:Novartis Pharmaceuticals Corporation, Turkey: Employment. Dag:Novartis Pharmaceuticals Corporation, Turkey: Employment.

The Linker Protein GAS7 Negatively Regulates Pre-B Cell Differentiation and Amplifies Proliferation and Survival Signals in Acute Lymphoblastic Leukemia

Background:Growth arrest-specific gene 7 (Gas7) functions as an adaptor for SH2- and SH3-containing proteins, in particular in cells that undergo growth arrest. Gas7 is abundantly expressed in the brain and is involved in neuronal differentiation. Interestingly, MLL-GAS7 fusion molecules resulting from the t(11;17)(q23;p13) chromosomal translocation have been reported in treatment-related acute myeloid leukemia (AML; Megonigal et al., 2000) and in a pediatric acute lymphoblastic leukemia (ALL). While the function of MLL has been extensively studied, the role of its fusion partner GAS7 in normal hematopoiesis and leukemia has not been elucidated.Results: Studying gene expression changes during normal B cell development, we identified Gas7 as the gene with the strongest relative increase at the pre-B cell receptor checkpoint. At the transition from IL7-dependent Fraction C’ to IL7-independent small resting pre-B cells (Fraction D), GAS7 mRNA levels were upregulated by >13-fold in both human and mouse B cell progenitors. Withdrawal of IL7 cytokine signaling and Cre-mediated conditional deletion of Stat5ab recapitulated the strong increase of GAS7 expression under cell culture conditions. These finding suggest that GAS7 is part of an adaptive response of differentiating pre-B cells to attenuation of cytokine/Stat5 signaling. Consistent with this scenario, we found that Gas7-/-pre-B cells undergo accelerated differentiation, including spontaneous Ig κ light chain gene recombination and loss of Stat5-signaling. Conversely, overexpression of GAS7, reduced responsiveness of pre-B cells to normal differentiation stimuli. These findings suggest that the linker molecule GAS7 is a negative regulator of pre-B cell differentiation.Likewise, we found that tyrosine kinase inhibitor treatment of human Ph+ ALL cells resulted in a strong increased of GAS7 expression, in parallel with loss of Stat5 function. To elucidate the function of Gas7 in B cell lineage leukemia, we transformed bone marrow pre-B cells from Gas7-/- mice with BCR-ABL1. Gas7 deficient Ph+ ALL cells showed decreased proliferation with reduced S phase and increased apoptosis. In agreement with effects of Stat5 on the sensitivity of Ph+ ALL cells against tyrosine kinase inhibitors (TKIs), Gas7 deficient Ph+ ALL cells showed massively increased susceptibility to Imatinib-induced apoptosis. In addition, absence of Gas7 caused loss of self-renewal capacity and failure to form colonies in methylcellulose assay. Co-immunoprecipitation experiments with flag tagged GAS7 in patient-derived Ph+ALL cells revealed that GAS7 physically interacts with STAT5 and retains STAT5-Y694 in an active conformation.Thereby, GAS7 can propagate even weak Stat5 activity and maintain residual cytokine or BCR-ABL1 oncogenic signaling in normal and malignant pre-B cells.Conclusions: Here show that GAS7 functions as an important positive regulator of Stat5 downstream of cytokine receptors in normal pre-B cells and downstream of BCR-ABL1 and other oncogenes in leukemia. Owing to the GAS7-dependent reinforcement of Stat5-dependent survival and proliferation signaling, normal and leukemic pre-B cells can survive periods of reduced cytokine/oncogene signaling. These findings suggest that the interaction interface between GAS7 and Stat5 represents a potential target for small molecule scaffolds and peptides. DisclosuresNo relevant conflicts of interest to declare.

Twelve-Month Cost per Responder (CPR) Analysis of Nilotinib and Dasatinib As Front-Line Therapy in Patients with Philadelphia Chromosome-Positive (Ph+) Chronic Myeloid Leukemia in Chronic Phase (CML-CP)

Introduction: Nilotinib and dasatinib, two second-generation tyrosine kinase inhibitors (TKIs), have been associated with higher rates of molecular response compared to imatinib in the treatment of newly diagnosed CML-CP patients based on the ENESTnd and DASISION trials. A published indirect comparison study of efficacy outcomes between nilotinib and dasatinib showed that nilotinib had significantly higher rates of molecular response by 12 months compared to dasatinib: major molecular response (MMR; defined as BCR/ABL1 level ≤0.1% on International Scale [IS]): 56.1% vs. 45.9%; MR4.0 (BCR/ABL1 level ≤0.01% on IS): 20.6% vs.12.0%; and MR4.5 (BCR/ABL1 level ≤0.0032% on IS): 10.2% vs. 3.1% for nilotinib and dasatinib, respectively. However, little is known about the costs associated with achieving molecular response. This study estimated and compared the CPR for the first 12 months of nilotinib and dasatinib for the treatment of Ph+ CML-CP in a front-line setting from a US payer’s perspective.Methods: A 12 month CPR model of nilotinib and dasatinib was constructed, where MMR by 12 months was used to define a responder and the rates were from the published indirect comparison study. The two model cost components were 12 month treatment costs and treatment-related grade 3/4 adverse event (AE) costs. Treatment costs were estimated based on drug price (wholesale acquisition cost), median dose and discontinuation rate. Drug prices by dose were extracted from the Red Book Online®. Median doses and discontinuation rates were from the ENESTnd, DASISION and the 2nd-line trials of nilotinib and dasatinib. Discontinuation was assumed to occur at a constant rate over the 12 months. Patients who discontinued the initial treatment were assumed to switch to the other second-generation TKI and remain on this for the rest of the first year. No patient was assumed to die within the first 12 months, i.e. no patients discontinued due to death. AE costs were estimated based on AE rates and the costs of treating the AE: treatment-related rates of grade 3/4 AEs were obtained from the clinical trials and direct medical costs were obtained from published literature. Because the definition of AEs may differ across the two trials, AE rates were adjusted relative to the AE rates observed in the imatinib arms of the two trials. The 12 month CPR for each treatment was calculated as the sum of the 1st-line treatment costs, the 2nd-line treatment costs (after a switch), and the grade 3/4 AE costs incurred by responders and non-responders, divided by the MMR rate. All costs were presented in 2014 USD. One-way sensitivity analyses varying the response definition (including MR4.0 and MR4.5) and AE costs were conducted.Results: The 12 month CPR was $202,840 for nilotinib and $248,919 for dasatinib (difference=$46,079; p <0.05). Costs of neutropenia, thrombocytopenia and anemia were the driving factor behind AE costs; these costs in the nilotinib arm were about half (53%) of the costs for of these AEs in the dasatinib arm. In one-way sensitivity analyses with responders defined based on MR4.0 and MR4.5, the differences in CPR between the two drugs were even greater: CPR for nilotinib was 42% and 70% (both p<0.05) lower than that for dasatinib for MR4.0 and MR4.5, respectively. In one-way sensitivity analyses that varied the costs of AEs to their lower and upper bounds, CPR ranged from $201,277 to $217,298 for nilotinib and $245,245 to $280,568 for dasatnib. Nilotinib’s CPR was 17.9% and 22.6% (both p<0.05) lower than dasatinib’s CPR for the low and high AE cost estimates, respectively.Conclusion: This analysis demonstrates that, compared to dasatinib, front-line therapy with nilotinib is associated with a lower CPR during the first 12 months in patients with newly diagnosed CML-CP from a US payer’s perspectiveAbstract 1304. Table 1CPR ResultsResponse Rates*Treatment CostsGrade 3 / 4 AE CostsCPRCPR Difference(Nilo – Dasa)NiloDasaNiloDasaNiloDasaNiloDasaMMR56.1%45.9%$112,024$110,901$1,769$3,353$202,840 $248,919 -$46,079 p <0.05MR4.020.6%12.0%$552,394$952,117-$399,723 p <0.05MR4.510.2%3.1%$1,115,619$3,685,613 -$2,569,994 p <0.05Nilo: Nilotinib; Dasa: Dasatinib*Signorovitch, J.E., et al. One-year and longer-term molecular responses to nilotinib and dasatinib for newly diagnosed chronic myeloid leukemia: a matching-adjusted indirect comparison. Abstract presented at American Society for Clinical Oncology, Chicago, May 30-June 3, 2014. DisclosuresGuerin:Novartis Pharmaceuticals Corporation: Consultancy, I am an employee of Analysis Group, Inc., which received consultancy fees from Novartis Other. Revol:Novartis Pharmaceuticals Corporation: Consultancy, I am an employee of Analysis Group, Inc., which received consultancy fees from Novartis Other. Ramanakumar:Novartis Pharmaceuticals Corporation: Consultancy, I am an employee of Analysis Group, Inc., which received consultancy fees from Novartis Other. Foster:Novartis Pharmaceuticals Corporation: Consultancy, I am an employee of Analysis Group, Inc., which received consultancy fees from Novartis Other. Wu:Novartis Pharmaceuticals Corporation: Consultancy, I am an employee of Analysis Group, Inc., which received consultancy fees from Novartis Other. Mhatre:Novartis Pharmaceuticals Corporation: Employment. Chen:Novartis Pharmaceuticals Corporation: Employment, Stock options Other.

The Effect of Nilotinib in Chronic Myeloid Leukemia Treatment Dose on Spermatogenesis and Folliculogenesis in a Healthy Mouse Model

Abstract Introduction&amp;Aim:Chronic myeloid leukemia (CML) is a hematopoietic pluripotent stem cell disease where myeloid cells lead to uncontrolled proliferation. Current treatment of Ph (+) CML is based on the inhibition of tyrosine kinase inhibitors (TKI), especially second generation drugs. Majority of CML patients are male and 46% of them are between 20 and 64 years of age. Therefore, it is conceivable that inhibition of c-kit or PDGFR by TKI may have deleterious effects on spermatogenesis or folliculogenesis, resulting in male or female subfertility. Aim of this study is to determine the effect of nilotinib on spermatogenesis and folliculogenesis which is used routinely to treat CML. Materyal&amp;Method: Here we present the results of testicular and ovarian changes after nilotinib administration to five-week old male and female C57bI6 mice. Mice received 0.4 mg of nilotinib per day dissolved in the drinking water for 2 months. Control group received only drinking water. Treatment dose was determined according to the clinical studies regarding the plasma concentrations (20 mg/kg, orally). After sacrification of both groups, testicular and ovarian tissues were fixed and parafin sections were stained with hematoxylene-eosin. In the ovaries, the follicles were counted and their developmental stages were recorded from the serial sections. In the testes, 24 seminiferous tubules with approximately circular cross-sectional profiles were assessed using a classification according to the degree of spermatogenic activity to generate a mean score for each mouse. In addition, tubule diameters were measured using an eyepiece micrometer to provide an additional indication of the level of function. Results:There was less distance between the cortex and medulla than normal, and the follicles were widely scattered instead of organized in a normal hierarchy from the least mature at the periphery to the largest growing stages towards the medulla. The numbers of follicles were significantly different between nilotinib and control groups (268±110 vs. 170±60; p= 0.03). Virtually every seminiferous tubule from all animals had active spermatogenesis with either spermatids or spermatozoa present. Mean tubular diameter measurements were 190,61±8,33 vs. 194,32±7,26 in control and nilotinib groups, respectively (p=0.475). Spermatogenic activity index were not significantly different between control and nilotinib groups (3.1 vs. 3.4; p=0.241). Conclusion: Unlike the manufacturer’s results; we showed the supression of folliculogenesis and prevention of spermatogenesis during the long-term nilotinib treatment. Our results indicate that nilotinib, within the dose of CML treatment regimen, may create gonadotoxicity and therefore its usage may be an indication for fertility preservation. In the second part of our ongoing study, we are investigating the effect of nilotinib on fertility and teratogenicity. Disclosures No relevant conflicts of interest to declare.

PF-114, a potent and selective inhibitor of native and mutated BCR/ABL is active against Philadelphia chromosome-positive (Ph+) leukemias harboring the T315I mutation.

Targeting BCR/ABL with tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Resistance attributable to either kinase mutations in BCR/ABL or nonmutational mechanisms remains the major clinical challenge. With the exception of ponatinib, all approved TKIs are unable to inhibit the 'gatekeeper' mutation T315I. However, a broad spectrum of kinase inhibition increases the off-target effects of TKIs and may be responsible for cardiovascular issues of ponatinib. Thus, there is a need for more selective options for the treatment of resistant Ph+ leukemias. PF-114 is a novel TKI developed with the specifications of (i) targeting T315I and other resistance mutations in BCR/ABL; (ii) achieving a high selectivity to improve safety; and (iii) overcoming nonmutational resistance in Ph+ leukemias. PF-114 inhibited BCR/ABL and clinically important mutants including T315I at nanomolar concentrations. It suppressed primary Ph+ acute lymphatic leukemia-derived long-term cultures that either displayed nonmutational resistance or harbor the T315I. In BCR/ABL- or BCR/ABL-T315I-driven murine leukemia as well as in xenograft models of primary Ph+ leukemia harboring the T315I, PF-114 significantly prolonged survival to a similar extent as ponatinib. Our work supports clinical evaluation of PF-114 for the treatment of resistant Ph+ leukemia.

Optimization of formulation and influence of environmental stresses on stability of lycopene-microemulsion

The stability of two-layer lycopene-microemulsions and the degradation of lycopene in microemulsions subjected to thermal processing and under environmental stress were investigated. The formulation of microemulsions made with different ratios of whey protein isolate (WPI), high-methylester-pectin (HMP), and an oil phase containing lycopene was optimized. The oil volume fractions, concentrations of WPI and HMP, and their interactions significantly influenced the physical stability of lycopene-rich microemulsions. The two-layer microemulsion consisted of WPC and HMP was much more stable under environmental stress compared to the WPC one-layer microemulsions. The one-layer lycopene-microemulsions destabilized at low pH, but the two-layer lycopene-microemulsion became unstable when the pH was in the neutral range of 6.12 to 7.01. The stability of microemulsions declined with increasing NaCl concentrations. About 36.8 g/100 g, 23.2 g/100 g, and 11.1 g/100 g of the total lycopenes were lost in the oil-phase, the one-layer lycopene-microemulsions, and the two-layer lycopene-microemulsions after thermal treatments, respectively. The optimized microemulsions contained 0.2 g/100 g (w/w) whey protein concentrate (WPC), 0.5 g/100 g HMP (w/w), and 5 mL/100 mL oil phase fraction had the highest physical stability. The optimized lycopene-microemulsion was more stable to thermal treatment and changes of pH, but become sensitive to NaCl treatment.

A multi-parameter in vitro screen in human stem cell-derived cardiomyocytes identifies ponatinib-induced structural and functional cardiac toxicity.

Ponatinib, a multi-targeted TKI and potent pan-ABL inhibitor, approved for the treatment of Ph + ALL and CML, was temporarily withdrawn from the U.S. market due to severe vascular adverse events. Cardiac-specific toxicities including myocardial infarction, severe congestive heart failure, and cardiac arrhythmias have also been shown with ponatinib. Targeted oncology agents such as ponatinib have transformed cancer treatment but often induce toxicity due to inhibition of survival pathways shared by both cancer and cardiac cells. These toxicities are often missed by the standard preclinical toxicity assessment methods, which include human Ether-à-go-go-related gene (hERG) and animal toxicity testing. In this study, we show that a multiparameter in vitro toxicity screening approach using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) accurately predicted the cardiac toxicity potential of ponatinib. This in vitro model evaluated ponatinib's effect on the overall cell health, mitochondrial stress, and function of hiPSC-CM and also provided mechanistic insight into the signaling pathways and cellular structures altered with treatment. We show here that ponatinib rapidly inhibits prosurvival signaling pathways, induces structural cardiac toxicity (as shown by actin cytoskeleton damage, mitochondrial stress, cell death, and troponin secretion), and disrupts cardiac cell beating. Most of these effects occurred at doses between 10× and 50× ponatinib's Cmax, a dose range shown to be relevant for accurate prediction of in vivo toxicity. Together these studies show that a comprehensive in vitro screening tool in a more relevant human cardiac cell model can improve the detection of cardiac toxicity with targeted oncology agents such as ponatinib.

Stromal cells attenuate the cytotoxicity of imatinib on Philadelphia chromosome-positive leukemia cells by up-regulating the VE-cadherin/β-catenin signal

β-Catenin is a key regulator of leukemia stem cell maintenance and drug resistance. Herein, we investigated the protective effects of the stromal cell-mediated VE-cadherin-β-catenin signal on Ph+ leukemia cells during imatinib treatment. We found stromal cells could desensitize imatinib and up-regulate VE-cadherin expression on Ph+ leukemia cells (K562 and SUP-B15 cells), which further stabilized and activated β-catenin. Knockdown of VE-cadherin with shRNA diminished the β-catenin protein and partly resensitized Ph+ leukemia cells to imatinib despite the presence of stromal cells, suggesting VE-cadherin is a potential target in the treatment of Ph+ leukemia.

Abstract 484: Identification of FOXM1 as therapeutic target in Philadelphia chromosome-positive acute lymphoblastic leukemia

Abstract Introduction: The BCR-ABL1 tyrosine kinase induces malignant transformation of B cells at the pre-B cell checkpoint and induces Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). In a genome-wide search, we identified FOXM1 as a transcription factor that is specifically upregulated at the pre-B cell checkpoint. FOXM1 is a forkhead box transcription factor and a key regulator of cell growth by promoting cell cycle progression. Results: We transformed murine pre-B cells with a retroviral BCR-ABL1 expression vector and observed upregulation of Foxm1 protein levels. Consistent with this, FOXM1 protein levels in patient-derived Ph+ ALL samples are ∼10-fold higher than in healthy B cells and B cell precursors (n=5; P=0.01). In a cohort of 83 Ph+ ALL patients, the FOXM1 promoter region was significantly de-methylated compared to normal pre-B cells (n=12; P=2.4x10e-7). In order to evaluate a potential predictive value of FOXM1 expression in ALL cells, we correlated FOXM1 mRNA levels with clinical outcome. High FOXM1 expression correlates with shorter overall survival in patients with ALL (n=12, P=0.05) and was predictive of a higher risk of relapse (n=21; P=7.3x10e-5). To study the functional role of FOXM1 in Ph+ ALL, we developed a genetic model for Foxm1 loss-of-function using Foxm1fl/fl mice. Cre-mediated, inducible deletion of Foxm1 decreases ALL cell viability (n=3; P=0.001) and reduced proliferative capacity (n=3; S-phase Foxm1+/+ 59% vs 42% for Foxm1-/-; P=0.0004). The ability to form colonies in vitro was significantly decreased by deletion of Foxm1 (n=3, P=8.9x10e-6). Injection of 1x10e5 Foxm1+/+ or Foxm1-/- leukemic cells into NOD/SCID mice revealed reduced leukemogenesis in vivo (n=7; P=0.0035). In contrast, normal B cell precursors were unaffected by Foxm1 deletion both, in vitro and in vivo. In addition, Foxm1-deleted ALL cells revealed a strikingly higher sensitivity towards Imatinib in dose-response curves compared to control cells (IC50=420 nM for Foxm1+/+ vs 160 nM for Foxm1-/-). Mechanistically, we found that FOXM1 decreased levels of reactive oxygen species via upregulation of the antioxidant response molecule catalase. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide (AA 26-44) and the natural occurring antibiotic Thiostrepton. Both bind and inhibit the function of FOXM1, and induced apoptosis in Ph+ ALL. Patient-derived Ph+ ALL cells were xenografted into NOD/SCID recipients and then treated with Thiostrepton i.v. for 7 consecutive days. Thiostrepton-treatment resulted in significantly prolonged overall survival (29 vs 35 days; n=7; P=0.0014) and reduced leukemia burden as measured by luciferase bioimaging, as compared to vehicle controls. Conclusion: Taken together, our data identify the pre-B cell-specific transcription factor FOXM1 as a novel therapeutic target for the treatment of TKI-resistant Ph+ ALL. Citation Format: Maike V. Buchner, Eugene Park, Lars Klemm, Huimin Geng, Dragana Kopanja, Pradip Raychaudhuri, Markus Müschen. Identification of FOXM1 as therapeutic target in Philadelphia chromosome-positive acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 484. doi:10.1158/1538-7445.AM2014-484

Abstract 2447: Lineage-specific metabolic reprogramming in BCR-ABL1-driven leukemia

Abstract Background &amp; Hypothesis: The serine-threonine liver kinase B1 (LKB1) activates AMP-activated protein kinase (AMPK) and negatively regulates aerobic glycoloysis (Warburg effect). LKB1 and AMPK have long been established as tumor suppressors, leading to clinical trials that test the efficacy of AMPK activators as cancer therapeutics. Paradoxically, we found that high expression levels of LKB1 and subunits of AMPK at diagnosis correlate with poor clinical outcome in patients with high risk B precursor acute lymphoblastic leukemia (ALL) (n = 207). These findings seem to contradict the historical notion of LKB1-AMPK as a tumor suppressor pathway, suggesting that the functions of LKB1-AMPK pathway may depend on cellular and genetic contexts. Results: Here, we focus on the role of LKB1 in BCR-ABL1-driven leukemia - chronic myeloid leukemia (CML) and B cell lineage Ph+ ALL. To do so, genetic mouse models for 4-hydroxytamoxifen (4-OHT)-inducible deletion of Lkb1 in BCR-ABL1-transformed hematopoietic stem and progenitor cells (CML-like) and B cell progenitors (Ph+ ALL) were developed. In agreement with previous findings in solid tumors, Cre-mediated Lkb1 deletion in CML-like cells resulted in enhanced proliferation. Unexpectedly, deletion of Lkb1 in Ph+ ALL cells led to apoptosis and cell cycle arrest. Moreover, Lkb1deletion delayed the onset of Ph+ ALL development as well as prolonged overall survival of transplant recipient mice in vivo. Consistent with the above observations, Arf, p53 and p27 levels were reduced in Lkb1-deficient CML cells, while Lkb1 deletion in Ph+ ALL cells up-regulated Arf, p53 and p27 levels. Decreases in glucose consumption and lactate production were also observed in Lkb1-deificient Ph+ ALL cells; however, increases in the levels of glucose consumed and lactate produced were detected in CML cells following Lkb1 deletion. Importantly, inhibition of AMPK using Compound C (an ATP-competitive inhibitor) resulted in apoptosis in patient-derived Ph+ ALL cells, while Compound C had no significant effects on the viability of a panel of lymphoma and multiple myeloma cell lines tested. Furthermore, patient-derived Ph+ ALL cells were resistant to treatment with various AMPK activators (metformin, phenformin and AICAR). Finally, Compound C showed synergistic responses in combination with Imatinib and different PI3K/AKT inhibitors (BKM120, AZD5363 and GSK690693) in Ph+ ALL. In vivo, Compound C in combination with BKM120, a PI3K inhibitor, exerted significantly more potent inhibitory effect on leukemia progression than each agent alone, prolonging the overall survival of recipient mice. Conclusions: Taken together, our findings demonstrate that LKB1 plays divergent roles in myeloid lineage CML and B cell lineage Ph+ ALL. While AMPK activators were shown to be effective against CML cells in previous studies, inhibiting the LKB1-AMPK pathway may provide a better therapeutic avenue for treatment of Ph+ ALL. Citation Format: Lai N. Chan, Seyedmehdi Shojaee, Christian Hurtz, Huimin Geng, Carina Ng, Behzad Kharabi, Markus Müschen. Lineage-specific metabolic reprogramming in BCR-ABL1-driven leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2447. doi:10.1158/1538-7445.AM2014-2447

Physical Characteristics of Edible Film Gel Made under Various pH and Ratio of Casein and Tapioca

Gelation from casein and tapioca take place in the edible film making. An interaction of carbohydrate and protein in the formation of gel could be affect on the physical properties of edible film gel. An appropriate ratio of casein-tapioca and pH was expected to produce physical characteristics of edible film gel. The aims of this research were to know the influence of casein- tapioca ratio, pH and the interaction of casein-tapioca ratio and pH to produce edible film gel. The research was conducted by randomized completely block design with two factors. The first factor was casein-tapioca ratio (0:100; 20:80; 40:60; 60:40; 80:20; 100:0) and the second factor was pH (4; 7; 9). The parameter of observation were colour (lightness and chroma), moisture content, WHC, precipitation and viscosity of edible film gel. The data analysis uses minitab V.1.6 carried on Tukey test. The result shows that casein-tapioca ratio influenced on colour (lightness and chroma), moisture content, WHC, precipitation and viscosity of edible film gel. The treatment of pH influenced on colour (lightness and chroma), moisture content, WHC and precipitation of edible film gel. An interaction between the ratio of casein-tapioca and pH influenced on colour (lightness and chroma), moisture content, WHC and precipitation of edible film gel. Keywords: Edible film gel, interaction of carbohydrate-protein, physical characteristics

Hematologic safety data of single-agent ponatinib from a phase I and phase II trial in Philadelphia chromosome-positive (Ph+) CML/ALL patients.

e18021 Background: Ponatinib is a tyrosine kinase inhibitor (TKI) indicated for the treatment of Ph+ chronic myeloid leukemia (CML) or acute lymphocytic leukemia (ALL) in the absence of other appropriate TKI therapies or presence of the T315I BCR-ABL mutation. Hematologic toxicity can result in dose reductions, interruptions and discontinuation of drug therapy. In the following analysis, we summarize the hematologic safety profile of single agent ponatinib reported in 468 patients from 2 trials. The primary objective is to evaluate the number and percent of patients with hematologic toxicities based on previously published data. Methods: Data reported from patients treated with ponatinib in trials 10-201 and 07-101 was included in the analysis. Ponatinib was dosed at 45 mg orally once daily. Hematologic adverse effects analyzed included thrombocytopenia, neutropenia, anemia and arterial thrombotic events. Results: See Table below for summary of events. Thrombocytopenia, neutropenia and anemia were more fr...

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Bosutinib for the Treatment of Ph+ Chronic Myelogenous Leukemia. Where Does It Fit?

Bosutinib for the Treatment of Ph+ Chronic Myelogenous Leukemia. Where Does It Fit?

Profile of imatinib in pediatric leukemia

Profile of imatinib in pediatric leukemia Michael J BurkeDepartment of Pediatrics, Division of Hematology/Oncology/Bone Marrow Transplantation, Medical College of Wisconsin, Milwaukee, WI, USAAbstract: Using targeted therapy for treatment of cancer has become the paradigm to which clinical trials aspire. Imatinib, the BCR-ABL1 tyrosine kinase inhibitor (TKI), was the first of its kind to specifically target and inhibit the underlying Philadelphia chromosome (Ph+) oncogene found to be driving chronic myeloid leukemia in adults, and has since become standard of care for the treatment of chronic myeloid leukemia in children. Imatinib, with its ability to target Ph+ leukemia, has been successfully incorporated into the treatment of not only pediatric chronic myeloid leukemia but also Ph+ acute lymphoblastic leukemia. With the incorporation of imatinib into combination chemotherapy for pediatric Ph+ acute lymphoblastic leukemia, current survival rates are far higher than at any other time for this once dreadful disease. With more children today receiving treatment with imatinib for either chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia, knowledge is accumulating surrounding the short-term and long-term toxicities observed in children, adolescents, and young adults treated with this TKI. In summary, the TKI imatinib has made a historic impact in the treatment of pediatric Ph+ leukemias, transforming what were once very high-risk diseases with considerable morbidity and mortality into ones that are now very treatable but with a new awareness surrounding the long-term toxicities that may come with this price for cure.Keywords: imatinib, leukemia, lymphoblastic leukemia, chronic myeloid leukemia, pediatric

Imatinib in combination with allogeneic hematopoietic stem cell transplantation improved the outcome of adult Philadelphia chromosome-positive acute lymphoblastic leukemia

To investigate the role of imatinib in induction therapy for newly diagnosed adult patients with Philadephia chromosome-positive acute lymphoblastic leukemia (Ph⁺ALL), as well as the status of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of adult Ph⁺ ALL in imatinib era. Retrospectively analyzed 97 newly diagnosed adult Ph⁺ ALL patients from 2005 to 2013. According to whether administrated imatinib in the induction therapy and the administrating duration (≥3 d) , they were divided into imatinib (n=37) and non-imatinib group (n=60), and the former group was further divided into early-use (n=26) and late-use imatinib groups (n=11) (bounded by the fourteenth day of induction chemotherapy). We compared the overall response rate (ORR) and the negative rate of BCR-ABL fusion gene in patients who achieved complete remission (CR) or CR with incomplete recovery of blood cells (CRi) among the three groups at the end of the first induction therapy. There were 44 cases underwent allo-HSCT (transplant group) and 33 cases only adopted imatinib-based chemotherapy (non-transplant group) in 77 patients who administrated imatinib as a maintenance therapy, we further compared the incidences of overall survival (OS) , disease-free survival (DFS), relapse and nonrelapse mortality (NRM) between the two groups; and dynamically monitor polymerase chain reaction (PCR) negativity of patients who were in CR1 state before transplant (n=34) at the following timepoints of achieving CR or CRi, the first consolidation therapy, beginning the pretreatment of transplant and attaining hematopoietic reconstruction after transplant. After the first induction therapy, the ORR of imatinib group was significantly higher than of non-imatinib group (97.3%, 72.9% respectively, P=0.002), but early-use and late-use imatinib groups had no statistical significance in ORR (100% , 90.9% respectively, P=0.297); the rate of negativity of imatinib and non- imatinib groups were 20.0% and 0 respectively (P=0.041) in patients who achieved CR or CRi. The negative rate of patients in CR1 state before transplant attained 20.8%, 42.3%, 51.8%, 76.8%, respectively at the previously described 4 timepoints. And the differences between the fourth and the third, the third and the first timepiont all reached statistical significance (P=0.044, 0.022, respectively). The 5-year OS of transplant and non- transplant groups showed statistical difference (47.0%, 28.0% respectively, P=0.016), also for 5-year DFS (P=0.001) and the cumulative rate of relapse (P=0.000) of the former surpassing the latter; the cumulative rate of NRM between these two groups had no statistical significance (P=0.370). Conventional induction chemotherapy in combination with imatinib in the first induction therapy of adult Ph⁺ ALL, not only improved the rate of hematologic remission, also the rate of molecular response. Imatinib used as a consolidation and maintenance therapy after remission, and allo-HSCT scheduled as soon as possible improved the prognosis.

Ponatinib: a third-generation inhibitor for the treatment of CML.

The establishment of imatinib as the standard therapy for CML marked the beginning of a new era of treatment. Due to occurring intolerance and resistance against the drug, developing newer inhibitors was promoted. This led to the second-generation inhibitors dasatinib, nilotinib and bosutinib. Despite all achieved improvement, all first- and second-generation inhibitors are ineffective against the BCR-ABL T315I "gatekeeper" mutation. In order to overcome this issue and to further improve the inhibitory effect, the third-generation inhibitor ponatinib was developed. Various clinical trials have been launched to study the effect of ponatinib in the clinical setting. Based on positive phase 1 and phase 2 trials, ponatinib was approved for the second-line treatment of CML and Ph+ ALL in December 2012 in the United States and in July 2013 in the European Union. Further trials investigate the potential effect of ponatinib in kinase-dependent subgroups of other malignancies. In conclusion, ponatinib has proved to be a powerful BCR-ABL inhibitor, which exhibits clinical activity both in BCR-ABL wild-type and mutant CML, including activity against the T315I mutation. Despite previous TKI failure, chronic-phase CML patients can achieve sustained remissions using the novel drug, offering a new therapeutic option in the treatment for CML.

Dasatinib.

Dasatinib is an orally available short-acting dual ABL/SRC tyrosine kinase inhibitor (TKI). It potently inhibits BCR-ABL and SRC family kinases (SRC, LCK, YES, FYN), but also c-KIT, PDGFR-α and PDGFR-β, and ephrin receptor kinase. Dasatinib is an effective treatment for chronic myeloid leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). Both diseases are characterized by a constitutively active tyrosine kinase; BCR-ABL. Dasatinib inhibits BCR-ABL with greater potency compared with other BCR-ABL inhibitors and is active in CML resistant or intolerant to imatinib. Dasatinib is approved for the treatment of CML (all phases) and for the treatment of Ph+ ALL, resistant or intolerant to prior imatinib treatment. Randomized trial data in CML show that first-line dasatinib provides superior responses compared with imatinib and enables patients to achieve early, deep responses, correlated with improved longer-term outcomes. A once-daily dose of 100mg in chronic phase CML results in high hematologic and molecular remission rates and prolongation of survival. In accelerated and blastic phase of CML, as well as in Ph+ ALL, complete hematologic and cytogenetic remissions frequently occur. Remissions however are very short. In these patients, once-daily 140mg is the recommended dose. The effect of dasatinib in other malignancies including solid tumors is subject of clinical studies. Regardless of many clinical trials in different tumor types and in different combinations of dasatinib with other agents, the role of dasatinib in the treatment of solid tumors has not yet been defined. Side effects of dasatinib are frequent but mostly moderate and manageable and include cytopenias and pleural effusions. The review presents the preclinical and clinical activity of dasatinib with a focus on clinical studies in CML.

Inhibition of PI3K/mTOR Overcomes Nilotinib Resistance in BCR-ABL1 Positive Leukemia Cells through Translational Down-Regulation of MDM2

Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.

Outcomes Of Tyrosine Kinase Inhibitors In Combination With Various Treatments Regimens In Adult Patients With Philadelphia Chromosome Positive Acute Lymphocytic Leukemia

BackgroundPhiladelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) is characterized by t(9;22) and is associated with poorer outcomes. Ph+ ALL responds initially to conventional chemotherapy but overall survival (OS) is low at < 20% with no curative form of post-remission chemotherapy. Allogeneic hematopoietic stem cell transplantation (HSCT) in first remission has historically been the only curative treatment modality, yet only cures approximately 30% of patients, typically in younger patients (less than 60 years of age). The introduction of tyrosine kinase inhibitors (TKIs) that target the fusion protein Bcr-ABL have altered the management of Ph+ ALL, with durable remission of 40-50%. Debate remains over the optimal chemotherapy “backbone” to combine with TKIs in the treatment of Ph+ ALL, given outcomes reported using nonmyeloablative approaches (Foà R, et al. Blood 2011; 118:6521). MethodsWe conducted a single-center, retrospective analysis of patients with newly diagnosed Ph+ ALL treated with various induction regimens in combination with TKIs. Patients aged 18 or older were included and comparisons were made between regimens considered to be intensely myelosuppressive (IMS) (i.e. hyperCVAD or ALL-2) and regimens considered less myelosuppressive (LMS) (i.e. ECOG 2993, L-20, or vincristine ± corticosteroids). The primary endpoint was a comparison of overall survival between treatment groups. Secondary endpoints included assessment of complete remission (CR) rates, disease free survival (DFS), likelihood to proceed to allogeneic HSCT, the effect of HSCT on overall survival, and hematologic and non-hematologic toxicities. ResultsTwenty-seven patients were included in this analysis. In combination with imatinib or dasatinib, eighteen patients were treated with IMS regimens while nine patients received LMS regimens. The groups were well-matched in baseline characteristics other than age (median: IMS - 49.6 years old vs. LMS -70.6 years old, p = 0.022). CR rates were high in both treatment groups (IMS - 94.4% vs. LMS -88.9%). No difference was observed in overall survival between the groups (3-year OS rate: IMS - 43.2% vs. LMS - 44.4%, Log-rank p = 0.921). There was a nonsignificant difference in DFS for patients favoring with IMS regimens (3-year DFS rate: IMS -43.2% vs. LMS - 33.3%, Log-rank p = 0.606). Patients treated with IMS regimens were more likely to proceed to transplant in CR1 [IMS - 88.2% (n = 15/17) vs. LMS - 22.2% (n = 2/ 9), p = 0.002). When evaluating the impact of HSCT on 3-year OS using a landmark analysis of 6 months post-induction therapy there was no difference between 13 patients who proceeded to HSCT compared to the 9 who did not (3-year OS rate post landmark: Yes HSCT - 33.7% vs. No HSCT - 55.6%, Log-rank p = 0.4). Overall, the toxicity during induction was similar in both treatment groups despite the increased myelosuppression that was expected in the more intense treatment group (100% experience grade 3 or greater hematologic toxicity vs. 89.9%). Among patients who achieve ANC>1000, those receiving LMS regimens did have a decrease in time to achieve this benchmark, though statistically it was not significant (median: 16 days vs. 21 days, p = 0.243). ConclusionWhile limited by the small sample size, it appears that the combination of TKIs with either IMS or LMS treatment regimens will achieve high CR rates. In our study OS was improved compared to historical controls prior to the introduction of TKIs and this includes a number of patients who did not undergo HSCT (Fielding AK, et al. Blood 2009; 113:4489-4496). It appears that in older patients who receive LMS regimens may not have inferior outcomes compared to younger patients who received IMS despite being far less likely to undergo HSCT in CR1. This finding may potentially prove to be significant given that up to 50% of patients with ALL older than 60 years of age are Ph+. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.