ALBERT

Description: Bim, a proapoptotic, BH3-only, Bcl-2 family member, is the major physiological antagonist of the antiapoptotic Bcl-2 proteins in B and T lymphocytes. It is essential for the induction of apoptosis of activated T cells following an immune response and for the homeostasis of B cells; it also plays a key role in the induction of apoptosis of early hematopoietic progenitors following cytokine-deprivation. We performed a CpG Islands prediction analysis on Bim promoter, identifying a putative CpG Island. Using a Bisulfite Modification-Clonal Sequencing Analysis (BMCSA), we investigated the methylation status of 19 CpG sites (from nucleotide −504 to +64 from the ATG start site) in the Bim promoter in 12 malignant hematological cell lines: 7 of lymphoid and 5 of myeloid origin. A minimum of 6 clones were analysed. An homogeneous, very high level of methylation was present in all the lymphoid cell lines (Average Level of Methylation (ALM) 93.4 ± 4.4% Standard Deviation [SD]) and a variable level of methylation in the myeloid cell lines (ALM 37.1 ± 32.4%). The lowest ALM was found in lymphocytes from healthy donors (15.5 ± 2.1%). Evidence of Bim promoter methylation was also found in frozen tumor samples from patients affected by NPM/ALK+ lymphomas. We treated the 12 cell lines with the demethylating agent 5-azacytidine (AZA). The changes in the methylation status of Bim promoter were evaluated by BMCSA and the corresponding induction of Bim by Real-Time PCR (TaqMan) and by Western Blot. The demethylation of Bim promoter led to a potent induction of Bim at the mRNA and protein level. In the lymphoid, NPM/ALK positive, SUDHL-1 cell line, in which a complete demethylation (from 100% to 0%) was achieved, the increase in the expression of Bim was 7.7-fold and this correlated with a potent induction of apoptosis, as assessed by TUNEL and Annexin V assays. Similar results were obtained using a different demethylating agent: 5-aza-2′deoxycytidine (DAC). To assess whether the methylation of Bim promoter is an active process, a wash-out experiment was performed on the SUDHL-1 (high level of methylation, 100%), on the PML/RAR alpha positive myeloid NB4 (intermediate level of methylation, 33%) and on the BCR/ABL positive LAMA-R cell lines (unmethylated) previously treated with AZA or DAC. This experiment showed that the demethylation is reversible and that, following remethylation, the expression of Bim at mRNA and protein level is reduced to the initial value. In the NB4 cell line, in which methylation is clustered on the last 6 CpG sites, remethylation occurs following the same pattern. No de novo methylation was seen in LAMA-R after the wash-out. To address the biological role for the methylation of Bim promoter, we generated a TET-ON inducible system for BimS (the most potent proapoptotic isoform of Bim) in the highly methylated NPM/ALK+ Karpas-299 cell line, showing that, following an induction of Bim expression, the cells are potently induced to apoptosis, as assessed by FACS using TUNEL and Annexin V assays. We conclude that Bim promoter is actively methylated in several leukemias/lymphomas of T and B origin and that its methylation is associated with the downregulation of Bim expression and with protection from apoptosis.

Description: Background Imatinib is effective in L-CP CML patients (Pts), but response duration is unknown. From 12/1999 to 05/2000, the Novartis study STI571A110 recruited 454 Pts with confirmed diagnosis of CP CML. Pts were hematologically (n=133) or cytogenetically resistant/refractory (n=160) or intolerant (n=161) to IFN. Median time since diagnosis was 34 months (m). Pts were evaluated for best major and complete cytogenetic response (MCyR and CCyR), time to progression to accelerated phase (AP) or blast crisis (BC), and overall survival (OS). This abstract includes data up to 56 m after the first patient and more than 48 m after last patient started treatment. Results: Median drug exposure as of 31-Jul-04 was 50 m. The initial daily dose was 400 mg. Dose increases after a median of 14 m were reported in 227 (50%) Pts. The table below summarizes discontinuation reasons, best responses observed and the estimated long-term outcomes at 48 m. n=454 (%) [95% Conf intervals] Still on Treatment 269 (59.3) Discontinued 185 (40.7) Progression/deaths from any cause 105 (23.1) AEs/toxicities 32 (7.0) BMT 5 (1.1) Withdrew consent/Lost/Admin. problems 43 (9.5) Pts achieving MCyR (includes CCyR) 301 (66.3) Pts achieving CCyR 248 (54.6) % Pts free of progression to AP/BC at 48 m (74.6) [70.4–78.9] OS at 48 m (82.4) [78.9–86.0] Median time to CCyR was 8.3 m [8.3–8.6 m]; 45 of the 248 Pts achieved CCyR after dose increase. The yearly risk of progression to AP/BC did not increase when considering all patients (7.8%, 6.0%, 7.2% and 7.1% within year 1,2,3,4 respectively). The corresponding risk rates for patients in MCyR at 3 m were 2.7%, 0.7%, 2.2%, 2.4%. Using the 3-m (and 12 m) landmark (n=446 or 421), Pts with CCyR, PCyR and minor CyR had an estimated OS at 48 m of 94(94)%, 93(94) %, 86(92) %, whereas Pts with only minimal CyR or no CyR had estimated 4-year OS of 85(78) % and 77(71) %, respectively. Among Pts who achieved MCyR and arrived to 48m of follow-up, 79% [74–84] maintain it; this value compares with 86% [80–92] for Pts with MCyR within 3 m. The following figure shows the duration of MCyR according to the time when MCyR was reached. Pts who had MCyR by the 3rd m did significantly better than the other groups (p= 0.02). However a MCyR obtained at a late time point (〉12 m) has a duration similar to the entire cohort. Half of the Pts who lost MCyR did not progress to AP while on study. Conclusion: Imatinib treatment for L-CP CML Pts failing IFN therapy is effective and produces durable cytogenetic responses and survival, with more than 80% Pts alive at 48 m and no evident increase in progression rate over time. Even Pts who failed to achieve any CyR reached OS of 71%. Obtaining a MCyR at 3–12 m resulted in 〉90% OS. Duration of MCyR was longer in Pts who responded within 3m, than in the other groups. The results will be updated for the meeting including 60-m data up to 31-July-05. Figure Figure

Description: The treatment of Chronic Myeloid Leukemia (CML) has been radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion protein Bcr-Abl, the cause of the disease. A variable portion of CML patients experience resistance to IM therapy. Resistance can arise from different mechanisms but in the vast majority of cases is due to point mutations into the protein sequence that alter directly or indirectly the drug-protein binding. Mutation sites can be schematically clustered in four region: the P loop, the IM binding site, the catalytic domain and the activation loop (A loop). At present more than 70 mutations conferring different levels of resistance have been found in CML patients. Recently, several new inhibitors have been developed in order to obtain an increased potency and a broad range of activity against IM resistant mutants. Nilotinib (NIL) is an IM derivative about 30-fold more potent than IM. Dasatinib (DAS) is a dual-specific Src/Abl inhibitor, structurally unrelated to IM and characterized by an activity 100 to 300-fold higher than IM. Bosutinib (BOS) is a dual Src/Abl inhibitor that shows an activity 10 to 30-fold higher than IM. It is known that resistance to second generation TKIs can also arise and the analysis of mutation profiles reveals substantial differences among different TKIs. Presently the choice of a TKI to treat a patient resistant to IM is mostly based on an empirical basis, e.g. the fact that a certain patient has not been previously exposed to that particular TKI. The possibility to directly compare the different activities of TKIs against a given mutation is of remarkable importance in clinical practice. Such a tool could be used similarly to an antibiogram for bacterial diseases, guiding the choice of the most appropriate inhibitor for each patient. In our study, we investigated the activity of BOS, DAS, IM and NIL against a panel of 18 mutated forms of BCR/ABL chosen to cover the most common mutations found in patients. Stable Ba/F3 transfectant cell lines were generated and the TKIs antiproliferative activity was determined by tritiated thymidine incorporation assay. The relative IC50 increase over wild type BCR/ABL (Relative Resistance RR) was calculated. We classified the RR values in three categories: sensitive (RR≤2), resistant (between 2.01 and 10) or highly resistant (〉10) as presented in the table. IC50-fold increase (WT=1) Imatinib Bosutinib Dasatinib Nilotinib Parental 10.78 38.31 〉50 38.45 WT 1 1 1 1 P-LOOP L248V 3.54 2.97 5.11 2.80 G250E 6.86 4.31 4.45 4.56 Q252H 1.39 0.81 3.05 2.64 Y253F 3.58 0.96 1.58 3.23 E255K 6.02 9.47 5.61 6.69 E255V 16.99 5.53 3.44 10.31 D276G 2.18 0.60 1.44 2.00 C-Helix E279K 3.55 0.95 1.64 2.05 V299L 1.54 26.10 8.65 1.34 Active site T3151 17.50 45.42 75.03 39.41 F317L 2.60 2.42 4.46 2.22 SH2-contact M351T 1.76 0.70 0.88 0.44 Active site F359V 2.86 0.93 1.49 5.16 A-LOOP L384M 1.28 0.47 2.21 2.33 H396P 2.43 0.43 1.07 2.41 H396R 3.91 0.81 1.63 3.10 G398R 0.35 1.16 0.69 0.49 C terminal lobe F486S 8.10 2.31 3.04 1.85 Sensitive ≤2 Resistant 2.01–10 Highly resistant 〉10 (Updated table available online at http://www.dimep.medicina.unimib.it/en/staff_174.php?docente_id=32) Our study points out at the differences in the activity spectrum of the 4 TKIs against the 18 Bcr/Abl mutations considered. The activity pattern presented in this work will help to reach a rational and tailored therapy offering to physicians a tool to use the new TKIs in the most efficient way for their patients.

Description: The oncogenic fusion protein Bcr-Abl is the underlying cause of Chronic Myeloid Leukemia (CML), and it is present in up to 35% of Acute Lymphoblastic Leukemia (ALL). The discovery of a selective Abl inhibitor, Imatinib mesylate, has revolutionized the treatment of CML. Recently, several new inhibitors have been developed with the aim of increasing both potency and selectivity against Abl. Bosutinib (SKI-606, Wyeth) is a dual Src/Abl inhibitor that showed an in vitro activity in the low nanomolar range on several BCR-ABL positive cells and it is, at present, in phase II clinical trials. Bosutinib is devoid of activity against some known “off-target” kinases blocked by imatinib, such as PDGFR and c-Kit. In addition structural and modelling data attribute to Bosutinib the ability to bind Bcr-Abl in the intermediate/active conformation, while Imatinib is able to bind only the inactive conformation of Bcr-Abl. In this study we analyzed in vitro the combination of Imatinib and Bosutinib in Bcr-Abl expressing cell lines to evaluate the possibility to decrease dosage of both drugs, increasing or maintaining the same efficacy but avoiding toxic effects. Combination effects were evaluated according to the method of Chou and Talalay, in which the combination index (CI) value is calculated for a combination of two drugs and allows the quantification of synergism: CI 1 indicate synergistic, additive or antagonistic interactions, respectively. Proliferation assays on a panel of Imatinib-sensitive and Imatinib-resistant BCR-ABL positive cell lines were performed. Cells were treated with Imatinib and Bosutinib as single agents or in three ratio combinations (1:3, 1:10, 1:33 in favour to Imatinib) across a wide range of concentrations. Combination indexes (CI) calculated at IC50, IC75 and IC90 for K562 cells (Imatinib-sensitive), suggest a synergistic to very strong synergistic effect (CI= 0.01-0.53). Similarly, in KCl22, KU812 and Lama84 cells (Imatinib-sensitive) moderate to strong synergistic effects were observed. A slight to moderate synergism was also obtained in three Imatinib-resistant cell lines tested: Lama84R (CI=0.63-0.88), K562R (CI=0.63-0.82) and KCL22R (CI=0.62-0.92). Western blot analysis of the tyrosine phosphorylation status of K562S cells treated with a mixture of 100nM Imatinib and 10nM Bosutinib revealed a substantially more pronounced inhibition compared with either 100nM Imatinib or 10nM Bosutinib alone. The effect of the combination was also assessed in murine Ba/F3 cells transfected with either wild type (WT) or mutated forms of BCR-ABL. Parental Ba/F3 cells were not affected by the presence of both drugs, while in Ba/F3 BCR-ABL WT the CI ranged from 0.49 to 0.85, indicating moderate synergism. The combination of Imatinib and Bosutinib inhibited the growth of Ba/F3 BCR-ABL Y253F with a slight synergism (CI 0.77-0.87). No synergistic effect was observed on Ba/F3 BCR-ABL E255K and on the highly resistant T315I mutant. Fresh leukemic cells obtained from three CML patients were also studied. In these samples synergistic effects between Bosutinib and Imatinib were confirmed (CI=0.52, 0.73, 0.62). The different binding modes of Imatinib and Bosutinib may justify the synergistic effect observed in the CML lines. This results support a possible therapeutic advantage for the combination of Bosutinib and Imatinib against Philadelphia positive leukemias.

Description: Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome positive (Ph1) acute lymphoblastic leukemia (ALL) are two fatal BCR/ABL-driven leukemias against which the current therapy with Abl kinase inhibitors fails to induce a long-term response, as the majority of patients are either refractory or relapse after a few months of treatment. We recently reported that functional loss of the PP2A tumor suppressor occurs during CML disease progression and that restoration of PP2A activity impairs in vitro and in vivo BCR/ABL leukemogenesis. Here we assessed the therapeutic potential of the PP2A activator FTY720 in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. FTY720 (500 nM-2.5 mM) induces caspase-dependent apoptosis (70–98% annexin V+) and impairs the clonogenic potential (70–95% inhibition) of imatinib/dasatinib-sensitive and -resistant (T315I) p210 and p190 BCR/ABL-expressing myeloid and lymphoid progenitor cell lines (Ph1 K562, 32D-p210BCR/ABL, 32D-p210(T315I)BCR/ABL and BaF3-p190BCR/ABL), respectively, and of primary bone marrow CML-BCCD34+ (n=11) and Ph1 ALLCD34+/CD19+ (n=12) patients cells. Interestingly the cytokine (IL-3 or IL-7)-dependent growth and differentiation of normal CD34+ myeloid and CD34+/CD19+ lymphoid progenitors (n=8) is not affected by FTY720 treatment. Furthermore, pharmacologic doses of FTY720 markedly suppress leukemogenesis in SCID mice (n=13 per group) transplanted with myeloid and lymphoid progenitor cells transformed with p210BCR/ABL and p190BCR/ABL, respectively. In fact, the median survival has not yet been reached in FTY720-treated (10 mg/kg/day) BCR/ABL+ cell-injected mice. Conversely, all of untreated 32D-p210BCR/ABL, 32D-p210BCR/ABL(T315I) and BaF3-p190BCR/ABL leukemic mice died of an overt acute leukemia-like process with a median survival of 4.3, 4.8 and 4.1 weeks, respectively (P

Description: Introduction:SIMPLICITY (NCT01244750) is an ongoing observational study of CP-CML pts in routine clinical practice receiving first-line (1L) imatinib (IM) prior to 2010 (retrospective) or 1L IM, dasatinib (DAS) or nilotinib (NIL) since 2010 (prospective) in the US and Europe. Methods: This analysis was designed to identify baseline characteristics associated with TKI switching in pts pooled from the prospective and retrospective cohorts. Pts were characterized as 'early switchers' (who discontinued 1L TKI and switched to a second-line [2L] TKI within 3 months of TKI initiation) or 'late switchers' (who discontinued 1L TKI and switched to a 2L TKI between 3 and 12 months after TKI initiation). A comparator group of 'non-switcher' pts did not discontinue 1L TKI within 12 months of TKI initiation; 'early non-switchers' did not switch 1L TKI within the first 3 months and 'late non-switchers' did not switch 1L TKI between 3 and 12 months after TKI initiation. Logistic regression analysis was used to identify factors associated with early vs. late switching (gender, age at diagnosis ≥65 years, 1L TKI, region, reason for discontinuation). Results: By March 07 2016, 1494 pts were enrolled at 197 sites in 7 countries. Within 12 months of initiating 1L TKI 238 pts (16%) had switched to 2L TKI. Of switchers, 31% and 69% were early and late, respectively. Of the overall cohort, 1189 pts were early non-switchers and 1146 were late non-switchers. Most early switchers received IM as 1L TKI (49%), followed by NIL (32%) and DAS (19%); most late switchers received IM (56%), followed by DAS (26%) and NIL (18%). Fewer early non-switchers than switchers received IM as 1L TKI (43%), followed by NIL (28%) and DAS (28%); fewer late non-switchers than switchers received IM (44%), followed by DAS (28%) and NIL (28%). Most switchers were female; a higher proportion of female switchers was early vs. late (63% vs. 52%, respectively). In comparison, 43% of pts in both non-switching groups were female. Median ages of early and late switchers were 54 (interquartile range [IQR]: 46-68) and 58 (IQR: 46-69) years, respectively; median ages of early and late non-switchers were 56 (IQR: 46-68) and 56 (IQR: 45-67) years, respectively. Logistic regression analysis showed that pts who switched due to intolerance had greater odds of switching early compared with those who switched due to resistance (odds ratio = 3.49; 95% confidence interval: 1.11-10.98; p=0.0323). Among the 238 pts who switched 1L TKI, the main reason for 1L TKI discontinuation was intolerance (early switchers: 76%; late switchers: 71%; Figure). The most common intolerances leading to changes in early and late switchers varied by 1L TKI. Most common for IM early switchers (n=26) was general disorders/administration site conditions (including fatigue, edema, pain) and skin/subcutaneous disorders; for IM late switchers (n=53), gastrointestinal (GI) disorders, general disorders/administration site conditions and skin/subcutaneous disorders. Most common for DAS early switchers (n=10) was GI disorders; for DAS late switchers (n=36), respiratory/thoracic/ mediastinal disorders. Most common for NIL early switchers (n=19) was GI disorders; for NIL late switchers (n=25), GI disorders, general disorders/administration site conditions and skin/subcutaneous disorders. TKI resistance less commonly contributed to 1L TKI discontinuation. Primary and acquired resistance were seen in higher proportions of late than early switchers (Figure). In late switchers, primary resistance was a more common reason for discontinuation in IM pts vs. DAS pts or NIL pts (22%, 5% and 4%, respectively). Acquired resistance was only reported as a reason for discontinuation in IM-treated pts (early switchers: 0.3%; late switchers: 11%). Conclusions: Of pts switching or discontinuing 1L TKI in the first 12 months of therapy, around one third switched within the first 3 months; compared with late switchers and non-switchers these pts were more likely to be younger and female. TKI intolerance was the main reason for switching and was significantly associated with early switching. Resistance was a less common reason for changing TKI therapy and was seen more frequently after 3 months and among pts treated with IM. More research is necessary to make definitive conclusions about comparisons between TKI groups, given the small numbers of pts in the TKI cohorts and the non-controlled nature of the trial. Disclosures Goldberg: COTA Inc: Employment; Novartis: Consultancy; Neostem: Equity Ownership; Pfizer: Honoraria; Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau. Michallet:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Astellas Pharma: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria. Hehlmann:Bristol-Myers Squibb: Consultancy; Novartis: Research Funding; German CML-Group: Research Funding. Zyczynski:Bristol-Myers Squibb: Employment. Foreman:ICON Clinical Research: Employment. Calimlim:ICON Clinical Research: Employment. Paquette:Incyte: Consultancy, Honoraria; Novartis: Consultancy; Ariad: Consultancy. Gambacorti:Pfizer: Honoraria, Research Funding. Cortes:ARIAD: Consultancy, Research Funding; Bristol-Myers Squib: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Zagorska:Bristol-Myers Squibb: Employment. Rong:Bristol-Myers Squibb: Employment. Mauro:BMS: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Novartis: Consultancy.

Description: Abstract 3763 Poster Board III-699 Bosutinib (Bos) (Wyeth Pharmaceuticals) is a dual Src-Abl tyrosine kinase inhibitor developed to inhibit the activity of BCR-ABL in chronic myeloid leukemia. P-glycoprotein (P-gp) is an ATP-binding cassette (ABC) transporter responsible of Imatinib (Im) efflux, and its increased expression can be related to Im resistance. The aim of the study was to define if P-gp is also responsible for the cellular efflux of Bos, and if P-gp altered expression can be related to Bos resistance. Four different K562 Ph+ cell lines were used. 1) K562S, Im sensitive; 2) K562DOX, cells resistant to Doxorubicin that overexpress P-gp (kind gift of JP Marie, Université Pierre et Marie Curie, Paris); 3) K562DOX siP-gp, K562DOX cells carrying a stable silencing of P-gp (kind gift of E Gunsilius, Innsbruck Medical University, Austria); 4) K562DOX H1, K562DOX cells carrying a control vector (kind gift of E Gunsilius). Real Time PCR confirmed that K562DOX and K562DOX H1 express higher levels of P-gp than K562DOX siP-gp (approximately 10 fold) and K562S (approximately 10000 fold). Western blot α-P-gp showed similar results. We then assessed Bos IC50 on the cell lines with a proliferation assay. K562DOX (IC50=175.3nM) and K562DOX H1 (IC50=102 nM) are resistant to Bos if compared to K562DOX siP-gp (IC50=9.1nM) and K562S (IC50=8.7nM). This data confirm that the P-gp overexpression is related to Bos resistance. Using C-14 radiolabeled Bosutinib (C-14 Bos) (Wyeth Pharmaceuticals) we set up an Intracellular Uptake and Retention (IUR) assay on K562S, K562DOX and K562DOX siP-gp cells. Cells were pre-treated for an hour with 0-60μM of Verapamil (Ver), a P-gp inhibitor, and then treated for two hours with 1μM C-14 Bos. In presence of Ver, K562DOX showed a significant increased amount of intracellular C-14 Bos ([] C-14 Bos Ver60μM/ [] C-14 Bos Ver0μM=5.8) that was not observed in K562S or K562DOX siP-gp. These data confirm that the intracellular concentration of Bos is strictly related to the expression levels and to the activity of P-gp. To test the biological effect of P-gp inhibition, we evaluated Bos IC50 by proliferation and apoptosis induction in K562DOX and K562S cells pre-treated with increasing concentrations of Ver. K562S did not show a significant decrease of Bos IC50 even at the highest concentration of Ver (22.5μM). In K562DOX the same concentration of Ver led to a 20 fold decrease of Bos IC50 (9nM), down to a level comparable to Bos IC50 in K562S cells (8.7nM). Thus, the inhibition of P-gp seems to restore the efficacy of Bos on proliferation inhibition and apoptosis induction in BCR-ABL+ cells. Finally, we evaluated the effect of P-gp inhibition on the phosphorylation levels of BCR-ABL with an α-phosphoTyrosine western blot. K562DOX and K562S cells were treated with Bos concentrations ranging from 0 to 80nM, and in absence or presence of Ver (5μM). Phosphorylation levels of BCR-ABL in K562S treated with Ver didn't show any difference when compared to untreated samples. In K562DOX, in absence of Ver, BCR-ABL was phosphorylated even in presence of high Bos concentration; the treatment with Ver restored the sensitivity to Bos, thus leading to a phosphorylation pattern similar to the one of K562S cells. This is compatible with increasing intracellular concentration of Bos after P-gp inhibition which results in increased molecular effect of Bos on BCR-ABL tyrosine phosphorylation. All together, these data confirm the relevance of expression levels and activity of P-gp for the intracellular concentration of Bos. The intracellular concentration of Bos is strictly related to its molecular activity on BCR-ABL and to its biological effects on Ph+ cells. Disclosures: Boschelli: Wyeth Pharmaceuticals: Employment.