Philadelphia Chromosome-positive Cells Research Articles

Characterization of Asciminib-Resistant Philadelphia Chromosome-Positive Cells.

Asciminib is approved for treating patients with chronic-phase chronic myeloid leukemia who were previously treated with two or more tyrosine kinase inhibitors or those with T315I mutation. However, the mechanisms underlying asciminib resistance remain unclear. In this study, we established a new asciminib-resistant cell line. We examined BCR::ABL1 gene mutation analysis and the effects of conventional chronic myelogenous leukemia inhibitors. Direct sequencing revealed Y139D and T315I mutations in asciminib-resistant cells. Ponatinib and omacetaxine were effective against asciminib-resistant cells. Y139D and T315I mutations are extremely resistant to asciminib. Ponatinib and omacetaxine show potential for treating asciminib-resistant chronic myeloid leukemia.

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.

Clinical characteristics, treatment pattern, and outcomes in newly diagnosed patients with chronic myeloid leukemia in the chronic phase by age

目的探讨初发慢性髓性白血病慢性期（CML-CP）不同年龄患者的临床特征、治疗和结局。方法回顾性分析2006年1月至2019年12月在北京大学人民医院确诊的≥14岁初诊酪氨酸激酶抑制剂（TKI）一线治疗的CML-CP连续病例。结果共收集957例患者，男性597例（62.4％），中位年龄40（14～83）岁。按年龄分为<40岁组（470例，49.1％）、40～59岁组（371例，38.8％）和≥60岁组（116例，12.1％）。随年龄增长，初诊时脾大（P<0.001）、WBC≥100×109/L（P<0.001）、贫血（P<0.001）、PLT<450×109/L（P＝0.022）、外周血原始细胞比例高（P＝0.010）和具有Ph染色体附加异常（P＝0.006）的患者比例降低，有合并症（P<0.001）、Sokal积分中/高危（P<0.001）和初始选择伊马替尼（P<0.001）的患者比例增高。而性别、ELTS危险度分布在各年龄组间差异无统计学意义（P值均>0.05）。多因素分析显示，≥60岁仅是影响患者总生存（OS）的不利因素（OR＝3.7, 95％ CI 1.5～9.2, P＝0.005），年龄与治疗反应和其他结局无显著相关性。TKI治疗中，随年龄增长，非血液学不良反应发生率显著增加（P<0.001），而血液学不良反应发生率相似。随访末期，随年龄增长，仍服用伊马替尼（P＝0.026）和服用减量TKI（P<0.001）的患者比例显著增加。结论初发CML-CP不同年龄组患者的临床特征、TKI用药选择和剂量、治疗反应、OS期和非血液学不良反应发生率存在差异。

Abstract IA17: Strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma

Abstract mTORC1 signaling is elevated in most lymphoid malignancies and frequently is associated with poor prognosis. Targeting mTORC1 with rapalogs or mTOR kinase inhibitors (TOR-KIs) are two strategies that both have limitations. Rapalogs are weak inducers of apoptosis, mainly due to incomplete mTORC1 inhibition, resulting in reduced phosphorylation of some substrates such as S6K1, but not 4E-BP1. TOR-KIs inhibit mTORC1 more completely, but also inhibit mTORC2, which likely contributes to dose-limiting toxicities in TOR-KI clinical trials. We have evaluated alternative strategies to target mTORC1 and downstream survival pathways in B-cell leukemia and lymphoma. In one approach, we have used novel bi-steric inhibitors of mTORC1, generated using our RevBlocksTM modular synthesis platform. These inhibitors engage both the FRB- and ATP-binding sites of mTOR and exhibit potent and selective inhibition of mTORC1. We found that RM-001, a proprietary bi-steric compound ~13-fold-selective for mTORC1 over mTORC2 in vitro, enhances the efficacy of the BCR-ABL1 inhibitor dasatinib in Philadelphia chromosome-positive pre-B cell acute lymphoblastic leukemia (Ph+ B-ALL) cell lines, causing profound and sustained phosphorylation inhibition of 4E-BP1 and S6, cell growth inhibition, and cell cycle arrest. Once-weekly dosing with RM-001 (10 mg/kg, IP) in a Ph+ B-ALL mouse model was well tolerated and effectively reduced leukemic burden as a single agent. A corresponding inhibition of phosphorylation of 4E-BP1 and S6 was observed in bone marrow. A key downstream effector pathway of mTORC1 is formation of the eIF4F translation initiation complex, which consists of eIF4E, eIF4G, and eIF4A. To assess targeting eIF4F as an alternative approach, we evaluated the function of eIF4F in both tumors and normal cells of the B lymphocyte lineage. In both B-ALL and diffuse large B-cell lymphoma (DLBCL) cells, disruption of eIF4F with a constitutively active 4E-BP1 mutant phenocopied the effect of TOR-KIs and bi-steric inhibitors. Notably, reduced gene dosage of Eif4e (eIF4E+/-) impaired the initiation and maintenance of B-cell transformation without affecting normal B-cell development and function. Most existing chemical inhibitors of eIF4F target either the cap-binding protein eIF4E or the RNA helicase eIF4A. We tested a novel eIF4G-binding molecule, SBI-756, shown previously to overcome resistance to BRAF inhibitors in melanoma models. SBI-756 (250 – 500 nM) disrupted eIF4F formation and caused marked cytotoxic effects in B-ALL, DLBCL, and normal B lymphocytes, while sparing T cells and NK cells. SBI-756 also synergized with targeted agents including dasatinib in Ph+ B-ALL and venetoclax in DLBCL. The combination of SBI-756 with venetoclax was more effective than single agents in a DLBCL xenograft model. These findings establish proof of concept for molecules that selectively bind to the scaffolding protein eIF4G. Collectively, these data highlight distinct strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma. Citation Format: Lee-or Herzog, Bianca J. Lee, Thanh-Trang Vo, Honyin Chiu, Sharmila Mallya, Amos Fung, Mallika Singh, James Aggen, Nidhi Tibrewal, Jacqueline A.M. Smith, David Wildes, Ze'ev Ronai, Davide Ruggero, David A. Fruman. Strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA17.

Novel compounds that target lipoprotein lipase and mediate growth arrest in acute lymphoblastic leukemia

Over the past decade, the therapeutic strategies employed to treat B-precursor acute lymphoblastic leukemia (ALL) have been progressively successful in treating the disease. Unfortunately, the treatment associated dyslipidemia, either acute or chronic, is very prevalent and a cause for decreased quality of life in the surviving patients. To overcome this hurdle, we tested a series of cylopropanecarboxamides, a family demonstrated to target lipid metabolism, for their anti-leukemic activity in ALL. Several of the compounds tested showed anti-proliferative activity, with one, compound 22, inhibiting both Philadelphia chromosome negative REH and Philadelphia chromosome positive SupB15 ALL cell division. The novel advantage of these compounds is the potential synergy with standard chemotherapeutic agents, while concomitantly blunting the emergence of dyslipidemia. Thus, the cylopropanecarboxamides represent a novel class of compounds that can be potentially used in combination with the present standard-of-care to limit treatment associated dyslipidemia in ALL patients.

Venetoclax Resistant Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Cells Are Also Resistant to ABL Inhibitors, Which Can be Overcome By Targeting the PI3K/AKT/ mTOR Pathway

Introduction: Venetoclax is a BH3-mimetic shown to specifically inhibit BCL-2 and has activity in hematologic malignancies addicted to BCL-2, including Ph+ ALL. However, when used as a single agent resistance develops through upregulation as well as post-translational modifications of MCL-1 from a variety of different pathways. It remains unknown what effects if any BCL-2 resistance will have on ABL kinase inhibition. In these studies, we investigated whether acquired resistance to venetoclax affected kinase sensitivity in a pre clinical model of Ph+ ALL.Methods: A venetoclax resistant cell line (SUPVR2) was generated by progressively exposing the Ph+ ALL cell line SUPB15 to increasing levels of venetoclax, and then characterized to the parent cell line. Expression of both pro and anti apoptotic members of the BCL-2 family of proteins was assessed via immunoblot. Sensitivity of resistant cells was then compared to parental cells across a library of small molecule kinase inhibitors. Briefly, cells were incubated with a panel of 130 small molecule inhibitors at varying concentrations for 72 hours, and then assessed using the colorimetric MTS assay as an indirect measure of viability. Drug effectiveness was evaluated based on the IC50 of each drug on the panel against each cell line. SUPB15 and SUPVR2 cells were also exposed to increasing concentrations of dasatinib or venetoclax after treatment with 40 µM of siRNA targeting ABL-1, MCL-1 or a non-specific control (NS). Drug combinations were assessed by co-culturing resistant cells with varying concentrations of each drug in combination or as single agent, and then synergy calculations were perfromed using Calculsyn software.Results: The IC50 of the SUPVR2 cells to venetoclax was >100µM, compared to 10nM for SUPB15. Levels of MCL-1 were higher and BCL-2 were lower in SUPVR2 cells as compared to SUPB15, but no differences were seen in BCL-xL or the anti-apoptotic molecules BIM, BAX/BAK, PUMA or NOXA. When assessed with the full drug panel, SUPVR2 were found to have reduced sensitivity compared to SUPB15 to the ABL TKIs nilotinib (IC50 257.7nM vs 10.7nM ) and dasatinib (306.4nM vs 4.6nM) but not ponatinib (1.5nM vs 1nM). In contrast, SUPVR2 cells showed enhanced sensitivity to molecules that targeted the PI3K/mTOR pathway (rapamycin (0.94nM vs15.6nM), PP242 (39.4nM vs 72.5nM), INK-128 (40.6nM vs 91.0nM), BEZ235 (29nM vs 531.1nM) and PI-103 (71.3nM vs 123.3nM). Although SUPVR2 did still harbor the BCR-ABL transcript, siRNA inhibition of ABL had no impact on viability compared to SUPB15, suggesting that the SUPVR2 are no longer ABL-dependent. siRNA inhibition of MCL-1 expression restored sensitivity of the resistant cells to venetoclax, but only partially restored sensitivity to dasatinib. Co-incubation of cells with dasatinib and either rapamycin, PP-242 or PI-103 led to restoration of dasatinib sensitivity in a synergistic manner.Conclusions: Resistance to venetoclax in SUPB15 leads to concurrent resistance to ABL kinase inhibitors, which appears to occur via activation of an ABL-independent pathway. Although it is not clear whether this resistance will develop if both an ABL kinase inhibitor and a BCL-2 inhibitor are administered concurrently, our results indicate that inhibitors of PI3K/mTOR may prevent the emergence of venetoclax and ABL resistance. DisclosuresTyner:AstraZeneca: Research Funding; Aptose Biosciences: Research Funding; Seattle Genetics: Research Funding; Array Biopharma: Research Funding; Janssen Research & Development: Research Funding; Leap Oncology: Consultancy; Takeda Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Genentech: Research Funding; Inctye: Research Funding. Druker:Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Array: Patents & Royalties; Oncotide Pharmaceuticals: Research Funding; Novartis: Research Funding; Pfizer: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Curis: Patents & Royalties; BMS: Research Funding; AstraZeneca: Consultancy; Array: Patents & Royalties; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; Roche: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; D3 Oncology Solutions: Consultancy; Ambit BioSciences: Consultancy; Agios: Honoraria; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Lorus: Consultancy, Equity Ownership; Cylene: Consultancy, Equity Ownership; CTI: Consultancy, Equity Ownership; Pfizer: Patents & Royalties; Curis: Patents & Royalties.

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.

Lenalidomide Effectively Induces Philadelphia-Chromosome Positive ALL Cells into Apoptosis in Synergy with Tyrosine Kinase Inhibitor

[Background] Lenalidomide (LMD) has been effectively used for the treatment of hematologic malignancies such as multiple myeloma and 5q-MDS. Recently, LMD was shown to bind the celebron (CRBN) E3 ubiquitin ligase complex and degrade Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3) via ubiquitin-proteasome system in myeloma cells, which leads to decrease in expression of downstream targets such as IRF4 and c-Myc. The IKZF1 is an essential transcription factor for the development of all lymphoid lineages particularly in the development of B- cell progenitors (BCP), and its gene consists of 8 exons including exons 4-6 and exon 8 encoding DNA-binding and dimerization domains, respectively. Genomic heterozygous deletions in IKZF1 are found in ~15% childhood BCP-ALL and the most frequent deletions affect the whole gene or exons 4-7 resulting in haploinsufficiency or expression of a dominant negative isoform. The IKZF1 deletions are now a hallmark for refractory nature of BCP-ALL linking to an unfavorable clinical outcome, and the frequency of IKZF1deletion is remarkably high (~70%) in Philadelphia-chromosome positive (Ph+) ALL cases. It is thus of great interest to examine LMD's effects on Ph+ALL cells.[Materials mad Methods] A total of 7 Ph+ALL cell lines with IKZF1 deletions (4 in exons 4-7, 2 in exons 2-7, and 1 in exons 1-8) were used to examine LMD's effects. The synergic effects of LMD with tyrosine kinase inhibitor (TKI; imatinib, dasatinib) were also examined. The ex vivo studies using NOD/Shi-scid, IL-2Rgnull (NOG) mice model were performed.[Results] 1. We first performed [3H]-thymidine uptake assays in the presence or absence of 20µM LMD for 3-6 days, and found that thymidine uptakes were variably inhibited (8~65%) after LMD treatment and two of sensitive cell lines showed a marked inhibition in a dose and a time dependent manners of LMD (~75%). Of importance, their thymidine uputakes were further inhibited (~95%) by LMD in the presence of the imatinib in a dose dependent manner (0.05-0.5nM). 2. To access the inhibition of thymidine uptake, we performed alamarBlue cytotoxic assays using the sensiitive cell line KOPN57bi, and found that the cell numbers modestly decreased by LMD (20 µM) or imatinib (0.5nM) , and markedly decreased (up to 35% at day 4) by LMD+imatinib. In flow cytometric analysis, KOPN57bi treated with LMD or imatinib showed cell cycle arrest alone, while the cell line treated with LMD+imatinib showed a marked increase in the subdiploid (42.3%), Annexin V-positive (72.0%), and active caspase 3-positive (31.8%) populations at day 4 after trreatment. The pancaspase inhibitor Z-VAD-FMK completely inhibited induction of cell death (apoptosis) by LMD+imatinib. These results were also obtained by use of another TKI dasatinib instead of imatinib. 3. We examined changes in expression of IKZF1 on Western blot using anti-C-terminal IKZF1 antibody in KOPN57bi (deleted in exons 4-7) 24h after LMD and/or TKI treatment, and found that several alternative splicing forms translated from intact IKZF1 disappear by LMD, but expression of a dominant negative Ik6 isoform was not affected by LMD presumably due to a lack of the CRBN binding site (residue Gln 146:exon5) in Ik6 isoform. IRF4 was also down-regulated by LMD and further in the presence of TKI. 4. We performed ex vivo studies using NOG mice. After intravenous injection of KOPN57bi (2x106cells), mice were orally administrated with saline (control, n=4), LMD (30mg/kg/d, n=5), imatinib (150mg/kg/d, n=5) , or LMD+imatinib (n=4) for 10 days (day 0,1,2,3,4, 7,8, 9,10,and 11) and percentages of human CD45 positive population in bone marrow mononuclear cells were examined at day 14. Human CD45+ cells showed 29.7±4.3% (mean±SEM) and 30.3±1.5% in control and imatinib-treated mice, respectively, whereas they were significantly (p=0.0002) decreased to 2.1±0.4% in LMD-treated mice. Of importance, they were further decreased to 0.3±0.4% in LMD+imatinib treated mice (p=0.0051), suggesting that combined treatment of LMD and imatinib should be very effective in xenograft mice models.[Conclusion] LMD renderd Ph+ALL cells a marked cell cycle arrest by completely abrogating the remaining IKZF1 function, and effectively induced them into apoptosis in synergy with TKI. Orally administration of LMD and TKI might become a high QOL treatment option for the treatment of Ph+ALL patients particularly for those with a higher age and organ dysfunctions. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1145: Zhx2 promotes survival of B-cell acute lymphoblastic leukemia cell lines

Abstract Zhx2, a zinc finger containing transcription factor is frequently overexpressed in B cell malignancies including B cell acute lymphoblastic leukemia (B-ALL), however its role in development of B cell malignancies remains unknown. Previously, dysregulated activity of transcription factors due to mutation e.g. Myc or overexpression e.g. Sox11 has been shown to perturb B cell differentiation leading to malignant phenotypes, resistance to chemotherapeutics and genetic heterogeneity. Zhx2 is required for normal neuronal and erythropoietic differentiation and has been identified as a tumor suppressor in hepatocellular carcinoma. Furthermore, analysis of public databases shows that Zhx2 is highly overexpressed in several cell lines representing B cell malignancies including B cell acute lymphoblastic leukemia (B-ALL) and B cell lymphoma. Consistently, Zhx2 is also overexpressed in patient samples derived from different subtypes of B cell malignancies. Quantification of Zhx2 mRNA expression obtained from human donors showed a marked upregulation during transition from common lymphoid progenitor to Pro B-cell stage suggesting potential role in B cell differentiation. Here we report that Zhx2 is overexpressed in Philadelphia chromosome positive (Ph+) B-ALL and T-ALL cell lines as well as Ph- B-ALL cell line. Zhx2 silencing in BV-173 (Ph+ B-ALL) and CMLT1 (Ph+ T-ALL) showed enhanced apoptosis with low doses of Dasatinib, a dual Src/Abl kinase inhibitor. Furthermore, global gene promoter and gene expression analysis on Zhx2 silenced cells identified putative Zhx2 regulated genes including CREB and YY1. Both of these transcription factors have roles in B cell differentiation, survival, proliferation and are commonly deregulated in B cell malignancies. We propose that Zhx2 regulates a B cell differentiation program and that overexpression of Zhx2 could lead to blocked differentiation of B cell progenitors promoting the malignant phenotype. Therefore, Zhx2 could be exploited as a chemo-therapeutic target in B cell malignancies. Citation Format: Namit Sharma, Huayun Hou, Michael D. Wilson, Catherine Jane McGlade. Zhx2 promotes survival of B-cell acute lymphoblastic leukemia cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1145.

The treatment of CML at an environment with limited resources

Objectives: This article reviews clinical experiences in the treatment of chronic myeloid leukemia (CML) in an environment of limited resources. Methods: We reviewed recent publications on Pub med and abstracts from mayor congresses relevant to the disease. Results: CML is a hematological neoplasm observed more frequently in adults, regardless of their socioeconomic status. Until recently, available treatments improved patients’ quality of life but did not modify survival. It was not until interferon appeared that patients received a drug that reduced and even eliminated Philadelphia chromosome-positive (Ph+) cells. Discussion: With the start of the new millennium, the International Randomized Study of Interferon-α plus cytarabine versus STI571 (IRIS) trial demonstrated a dramatic improvement in survival by comparing imatinib versus interferon alpha plus cytarabine. The Food and Drug Administration (FDA) approved imatinib as first-line treatment for newly diagnosed CML in 2001 due to its outstanding effectiveness. Years later, three second-generation (dasatinib, nilotinib, bosutinib) and one third-generation (ponatinib) tyrosine-kinase inhibitors (TKIs) were developed and approved. These highly effective treatment options, however, are not affordable for many low-income patients. Additionally, the use of drugs that effectively treat but do not cure the disease has resulted in an important economic impact for patients and health care systems worldwide, especially those in developing countries. Imatinib is the least expensive and a very effective TKI in many low-income countries. Early allogeneic stem cell transplantation must be considered in the management of selected patients before CML transformation.

ATG7 regulates energy metabolism, differentiation and survival of Philadelphia-chromosome-positive cells

ABSTRACTA major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34+ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease.

Pulmonary Talcosis in an Immunocompromised Patient.

The first case of pulmonary talcosis or talc pneumoconiosis related to inhalation of talc during its extraction and processing in mines was described by Thorel in 1896. Pulmonary talcosis is most commonly seen secondary to occupational exposure or intravenous (IV) drug abuse and, occasionally, in excessive use of cosmetic talc. Based on literature review, there has been an increase in reported incidents of pulmonary talcosis due to various forms of exposure to the mineral. We report an 82-year-old man who is diagnosed with Philadelphia chromosome positive pre-B cell acute lymphoblastic leukemia (ALL) treated with palliative imatinib who presented with chronic hemoptysis and dyspnea shortly after his diagnosis. His symptoms were initially thought to be due to an infectious etiology due to his malignancy, immunocompromised state, and radiographic findings until high-resolution computerized tomographic (HRCT) findings showed a diffuse pulmonary fibrosis picture that prompted further questioning and a more thorough history inquiry on his exposure to causative agents of interstitial lung disease. Very often, patients do not recognize their exposure, especially in those whose exposure is unrelated to their occupation. Our case emphasizes the need for thorough and careful history taking of occupational and nonoccupational exposure to known causative agents of interstitial lung disease.

Ribavirin Inhibits the Activity of mTOR/eIF4E, ERK/Mnk1/eIF4E Signaling Pathway and Synergizes with Tyrosine Kinase Inhibitor Imatinib to Impair Bcr-Abl Mediated Proliferation and Apoptosis in Ph+ Leukemia

The eukaryotic translation initiation factor 4E (eIF4E), which is the main composition factor of eIF4F translation initiation complex, influences the growth of tumor through modulating cap-dependent protein translation. Previous studies reported that ribavirin could suppress eIF4E-controlled translation and reduce the synthesis of onco-proteins. Here, we investigated the anti-leukemic effects of ribavirin alone or in combination with tyrosine kinase inhibitor imatinib in Philadelphia chromosome positive (Ph+) leukemia cell lines SUP-B15 (Ph+ acute lymphoblastic leukemia cell line, Ph+ ALL) and K562 (chronic myelogenous leukemia cell line, CML). Our results showed that ribavirin had anti-proliferation effect; it down-regulated the phosphorylation levels of Akt, mTOR, 4EBP1, and eIF4E proteins in the mTOR/eIF4E signaling pathway, and MEK, ERK, Mnk1 and eIF4E proteins in ERK/Mnk1/eIF4E signaling pathway; reduced the expression of Mcl-1 (a translation substrates of eIF4F translation initiation complex) at protein synthesis level not mRNA transcriptional level; and induced cell apoptosis in both SUP-B15 and K562. 7-Methyl-guanosine cap affinity assay further demonstrated that ribavirin remarkably increased the eIF4E binding to 4EBP1 and decreased the combination of eIF4E with eIF4G, consequently resulting in a major inhibition of eIF4F complex assembly. The combination of ribavirin with imatinib enhanced antileukemic effects mentioned above, indicating that two drugs have synergistic anti-leukemic effect. Consistent with the cell lines, similar results were observed in Ph+ acute lymphoblastic primary leukemic blasts; however, the anti-proliferative role of ribavirin in other types of acute primary leukemic blasts was not obvious, which indicated that the anti-leukemic effect of ribavirin was different in cell lineages.

Effects of stromal cells on sentitivity to imatinib in Sup-B15 Philadelphia chromosome positive acute lymphoblastic leukemia cells

目的探讨基质细胞OP9能否影响Ph+急性淋巴细胞白血病细胞株Sup-B15细胞对伊马替尼的敏感性以及作用机制。方法实验分为Sup-B15细胞组及基质细胞OP9与Sup-B15共培养组（即Sup-B15/OP9组）。采用CCK-8法测定细胞增殖抑制率；流式细胞术检测Annexin V/7-AAD标记的细胞凋亡率、CD34+CD38−细胞比例；荧光定量PCR检测细胞ALDH1、CD144、β-catenin mRNA水平；Western blot法检测细胞CD133、CD144、β-catenin蛋白表达水平；免疫共沉淀方法检测结合于CD144的β-catenin水平。结果10~45 µmol/L伊马替尼对Sup-B15、Sup-B15/OP9细胞均有增殖抑制作用，其IC50值分别为26.3和35.8 µmol/L，两组差异有统计学意义（P<0.05）。30 µmol/L伊马替尼作用于Sup-B15、Sup-B15/OP9细胞24 h，细胞总凋亡率分别为（14.24±2.11）%和（3.45±0.68）%，两组差异有统计学意义（P<0.05）。与Sup-B15组相比，Sup-B15/OP9组CD34+CD38−细胞比例增高[（3.42±0.28）%对（2.16±0.15）%，P<0.05]，ALDH1 mRNA水平增高（0.097±0.012对0.046±0.010，P<0.05），CD133蛋白表达水平升高；CD144 mRNA水平增高（0.103±0.015对0.010±0.003，P<0.05），CD144蛋白表达水平亦明显增高；β-catenin mRNA水平无明显变化（P>0.05），其蛋白总量明显上升，转位到细胞核的β-catenin蛋白明显增加，结合于CD144的β-catenin蛋白增加。结论与OP9细胞共培养后，Sup-B15细胞对伊马替尼敏感性下降，该作用可能与上调CD144表达、稳定CD144 /β-catenin信号通路、增加β-catenin核转位有关。

Combination of the ABL kinase inhibitor imatinib with the Janus kinase 2 inhibitor TG101348 for targeting residual BCR-ABL-positive cells.

BackgroundThe ABL kinase inhibitor imatinib is highly effective in treating most, but not all, patients with chronic myeloid leukemia (CML). This is because residual CML cells are generally present in the bone marrow microenvironment and are refractory to imatinib. Hematopoietic cytokine receptor signaling is mediated by Janus kinases (JAKs) and their downstream transcription factor, signal transducer and activator of transcription (STAT). TG101348 (SAR302503) is an oral inhibitor of JAK2.MethodsWe investigated the efficacy of imatinib and TG101348 using the break point cluster region-c-Abelson (BCR-ABL)-positive cell line and primary CML samples wherein leukemia cells were protected by a feeder cell line (HS-5).ResultsImatinib treatment resulted in partial inhibition of cell growth in HS-5-conditioned medium. Furthermore, combined treatment with imatinib and TG101348 abrogated the protective effects of HS-5-conditioned medium on K562 cells. Phosphorylation of Crk-L, a BCR-ABL substrate, decreased considerably, while apoptosis increased. In addition, the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased Crk-L phosphorylation, and increased apoptosis. We also investigated TG101348 activity against feeder cells and observed that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348.ConclusionsThese results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance of Philadelphia chromosome-positive cells.

Proteomic-based identification of Apg-2 as a therapeutic target for chronic myeloid leukemia

The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced “spontaneous” DNA damage and unfaithful repair in Philadelphia chromosome positive leukemia cells. Here, we investigated the changes of protein profile in H2O2-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially expressed and 13 proteins were identified with further MS analysis. Two essential proteins, Proto-oncogene tyrosine–protein kinase ABL1 (c-ABL) and Heat shock 70kDa protein 4 (Apg-2), were confirmed by Western blot and showed consistent changes with proteomic results. Moreover, functional analysis demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL, BaF3-BCR/ABLT315I). We also proved that Apg-2 inhibition aggravated H2O2 induced damage in BCR/ABL positive cells, and enhanced the sensitivity of BaF3-BCR/ABLT315I to STI571. Taken together, the findings in this work provide us with some clues to a better understanding of the molecular mechanisms underlying BCR/ABL in the DNA damage/repair processes and demonstrated that Apg-2 would be a valid target for anti-leukemia drug development.

Targeting autophagy potentiates tyrosine kinase inhibitor–induced cell death in Philadelphia chromosome–positive cells, including primary CML stem cells

Targeting autophagy potentiates tyrosine kinase inhibitor–induced cell death in Philadelphia chromosome–positive cells, including primary CML stem cells

Apoptosis and neutrophils in the regulation of Ph-positive myeloid cell proliferation and differentiation ex vivo

Ex vivo proliferation and differentiation of Philadelphia chromosome-positive (Ph+) human myeloid cells (Ph+ cells) from chronic myeloid leukemia (CML) proceed via alternation stages of cell proliferation and neutrophil maturation. To regulate them, apoptosis is alternately blocked or induced with the help of neutrophils and expression of bcr/abl, bax, and bcl2. The regulation of apoptosis in main types of Ph+ cells depends on the alternation of (1) Ph+ cell proliferation and (2) neutrophil maturation and may follow two pathways. One consists in alternating blockages and inductions of apoptosis with initial maturation and subsequent proliferation under alternation stages as (2)-(1)-(2) and has not been described as yet. Neutrophil accumulation blocks apoptosis. As neutrophils are depleted, apoptosis is induced again. Its block accelerates proliferation with a new accumulation of neutrophils, which is followed by regular neutrophil death and a new induction of apoptosis. The way optimizes the proliferation efficiency (P/D index) with a regular alternation of maturation and proliferation, allowing the cycle of proliferation and differentiation to be completed. In another way, the alternation starts with proliferation as (1)-(2)-(1) at a lower neutrophil content) and leads to resistant decrease of the maximal apoptosis level by a factor of 3–8 as compared with (2)-(1)-(2) alternation. A stable block of apoptosis is observed in cells with prolonged stages of proliferation and maturation, leading to an accumulation of blasts and myelocytes with elevated bcr/abl expression and expression of bcl2 > bax. A stable block of apoptosis is associated with CML progression and in Ph+ cell lines. Cells follow the first pathway of the apoptotic regulation in chronic-phase CML. Ex vivo cultivation of Ph+ cells from individual CML patients was assumed to provide for a more exact diagnosis of the CML phase and optimizing the treatment.

Comparison of Disease Outcomes for Patients with Chronic Myelogenous Leukemia in Chronic Phase Switched to Nilotinib or Dasatinib As Second-Line Therapy.

AbstractAbstract 2796 Background:Nilotinib and dasatinib are two tyrosine kinase inhibitors (TKIs) commonly used for the treatment of patients with chronic myelogenous leukemia in chronic phase (CML-CP) who are resistant or intolerant to imatinib. Both TKIs have been shown to be more potent than imatinib. However, no head-to-head clinical trials have been conducted comparing the efficacy of nilotinib to dasatinib as 2nd-line therapy for CML-CP patients resistant or intolerant to imatinib. This study compared disease outcomes of patients treated in the community who were switched from imatinib to 2nd-line nilotinib or dasatinib. Methods:Through an online chart abstraction form, participating community physicians submitted de-identified information on CML-CP patients ≥ 18 years old who switched after 10/28/2007 from imatinib to 2nd-line nilotinib or dasatinib with at least 30 days of follow-up. When multiple patients met the selection criteria, up to 8 patients in each group were selected randomly by physicians. Patients enrolled in clinical trials or with concurrent malignancies were excluded. Information was collected on demographics, clinical characteristics, cytogenetic response, progression to accelerated phase or blast crisis, and death from any cause. Documented complete cytogenetic response (CCyR) at a particular time point was defined as 0% Philadelphia chromosome positive cells on cytogenetic testing; patients with missing tests at that time point were counted as non-responders. Disease progression to accelerated phase or blast crisis, progression-free survival, and overall survival were compared using multivariate Cox proportional hazards models, adjusting for potential confounding factors, including patient demographics and clinical characteristics at the time the patient switched to a 2nd-line TKI. Results:122 hematologists and oncologists provided information on 597 patients, including 301 patients on nilotinib and 296 patients on dasatinib. The median follow-up was 11 months for nilotinib patients and 10 months for dasatinib patients. Age, sex, race, comorbidities, and other clinical characteristics were similar for both groups, except that nilotinib patients were more likely than dasatinib patients to have switched due to secondary imatinib resistance (p=0.047) and were less likely to have switched due to imatinib intolerance (p=0.024). Nilotinib patients had similar rates of documented CCyR compared to dasatinib patients at 12 months (37% vs. 35%, p=0.54), 24 months (42% vs. 40%, p=0.62), and 36 months (44% vs. 44%, p=0.95) following the switch. However, nilotinib patients had lower risk of progression (p=0.004), higher progression free survival (p=0.023) and a trend towards prolonged overall survival (p=0.067), adjusting for potential confounders. The table below summarizes these results. Conclusions:Among patients with CML-CP switched to nilotinib or dasatinib for 2nd-line therapy, nilotinib patients had significantly lower rates of progression and longer progression-free survival compared to dasatinib patients.OutcomeNilotinib EventsDasatinib EventsUnadjusted Hazard RatioP-valueAdjusted Hazard RatioP-value(n=301)(n=296)Progression5 (1.7)13 (4.4)0.39 (0.14–1.09)0.0720.15 (0.04–0.55)0.004Progression-free Survival286 (95.0)269 (90.9)1.81 (0.96–3.40)0.0672.14 (1.11–4.12)0.023Overall Survival291 (96.7)279 (94.3)1.71 (0.78–3.75)0.1772.13 (0.95–4.78)0.067 Disclosures:Griffin:Novartis: Consultancy, Research Funding. Guerin:Analysis Group, Inc.: Consultancy, Employment, I am an employee of Analysis Group, Inc, which has received consulting fees from Novartis Pharmaceuticals Other, Research Funding. Chen:Novartis Oncology: Employment, Own stock in Novartis Other. Macalalad:Analysis Group, Inc.: Consultancy, Employment, I am an employee of Analysis Group, Inc, which has received consulting fees from Novartis Pharmaceuticals Other, Research Funding. Luo:Analysis Group, Inc.: Consultancy, Employment, I am an employee of Analysis Group, Inc, which has received consulting fees from Novartis Pharmaceuticals Other, Research Funding. Wu:Analysis Group, Inc.: Consultancy, Employment, I am an employee of Analysis Group, Inc, which has received consulting fees from Novartis Pharmaceuticals Other, Research Funding.

Benefits of Early Switching From Imatinib to a Second-Generation Tyrosine Kinase Inhibitor Following 12 Month Complete Cytogenetic Response Failure: A Chart Review Analysis.

AbstractAbstract 2792 Background:The National Comprehensive Cancer Network (NCCN) guidelines (version 1.2013) recommend that patients with chronic myelogenous leukemia in chronic phase (CML-CP) should be tested for cytogenetic response 12 months following imatinib initiation. Failure to achieve complete cytogenetic response (CCyR) at 12 months should result in either an increased dose of imatinib (up to 800mg) or a change to a second-generation tyrosine kinase inhibitor (TKI). This study observed patients who failed to achieve CCyR at 12 months following the initiation of imatinib and compared treatment response rates and disease progression between patients who switched to a second-generation TKI early versus patients who did not. Methods:An online chart abstraction form was used to survey US oncologists and hematologists. Physicians submitted de-identified information on up to 10 adult patients with CML-CP who initiated imatinib as first line therapy (between 01/01/2007 and 26/07/2010) and failed to achieve CCyR at 12 months (between 10–14 months). Patients either switched to a second-generation TKI within 3 months following CCyR failure (early-switchers), or remained on imatinib for ≥3 months following CCyR failure (non-switchers). Non-switchers may have later switched to a second-generation TKI. The index date was defined as the date of the 12-month CCyR failure. Detailed patient information was collected, including demographics, comorbidities, imatinib dosage, and hematologic and cytogenetic response prior to the index date. Cytogenetic response and disease progression was also collected after the index date. CCyR was defined as 0% Philadelphia chromosome positive (Ph+) cells on cytogenetic testing. The proportion of patients achieving CCyR by 6, 12, and 24 months was reported among patients who had ≥1 cytogenetic test during these periods. Documented CCyR was defined as CCyR achievement analyzed among all patients, if patients were not tested for CCyR following the index date they were considered not to have achieved CCyR. Time to first documented CCyR achievement and time to disease progression were both estimated using multivariate Cox proportional hazard ratios (HR), where exposure was calculated from the index date to the first documented CCyR achievement, or to the date of progression, respectively. Multivariate regression analyses controlled for age, sex, race, index year, Charlson comorbidity index, imatinib dose and hematological response prior to index date, percentage of Ph+ cells and CML disease duration at index date, number of days between CML diagnosis and imatinib initiation, and rise in transcript level and chromosome abnormalities in Ph+ cells reported prior to the index date. Results:The majority of the 108 surveyed physicians were from a private practice (72.2%) and a small/intermediate practice size (61.1%). Physicians provided information on 593 patients who failed to achieve CCyR at 12 months; 306 were early-switchers and 287 were non-switchers. Among the non-switchers, 78 later switched to a second-generation TKI, and 104 increased imatinib dose after the index date. Patient demographics and comorbidities were similar among early-switchers and non-switchers, however, results of the 12-month cytogenetic test revealed that early-switchers had a greater number of Ph+ positive cells (51. 5 ± 16.6) compared to non-switchers (47.2 ± 13.1, p=.002). The median follow-up time was 612.5 days (range = 91–1625) and 591 days (range = 365–1623), respectively. Among patients tested for cytogenetic response during the follow-up period (274 early-switchers and 252 non-switchers), 35% of early-switchers subsequently achieved CCyR, compared to 24 % of non-switchers (p=.006). Within 6 months after the index date, 4.7% of the early-switchers achieved CCyR vs. 0.4% of non-switchers; by 12 months, 20.1% vs. 12.3% achieved CCyR; and by 24 months, 33.6% vs. 21.8% achieved CCyR, respectively (all p<.016). After adjusting for confounding factors, early-switchers had an 80% greater documented CCyR achievement rate compared to that of non-switchers (HR=1.80; p=.002) and a progression rate that was 81% lower (3.8% vs.1.5%, HR=0.19, p=.034). Conclusion:Early switching from imatinib to a second-generation TKI following 12-month CCyR failure was associated with better cytogenetic response and a lower risk of progression. Disclosures:DeAngelo:Novartis: Consultancy. Off Label Use: Everolimus in AML. Chen:Novartis Oncology: Employment, Own stock in Novartis Other. Guerin:Analysis Group, Inc.: Consultancy, Employment, I am an employee of Analysis Group, Inc, which has received consulting fees from Novartis Pharmaceuticals Other, Research Funding. Styles:Analysis Group, Inc.: Consultancy, Employment, Research Funding. Aberki:Analysis Group, Inc.: Consultancy, Employment, Research Funding. Giguere-Duval:Analysis Group, Inc.: Consultancy, Employment, Research Funding. Wu:Analysis Group, Inc.: Consultancy, Employment, I am an employee of Analysis Group, Inc, which has received consulting fees from Novartis Pharmaceuticals Other, Research Funding.