Constitutive Tyrosine Kinase Activity Research Articles

CCDC6: the identity of a protein known to be partner in fusion.

Coiled Coil Domain Containing 6 gene, CCDC6, was initially isolated as part of a tumorigenic DNA originated by the fusion of CCDC6 with the tyrosine kinase of RET receptor, following a paracentric inversion of chromosome 10. For a long time, CCDC6 has been considered as an accidental partner of the RET protooncogene, providing the promoter and the first 101 aa necessary for the constitutive activation of the oncogenic Tyrosine Kinase (TK) RET in thyroid cells. With the advent of more refined diagnostic tools and bioinformatic algorithms, an exponential growth in fusion genes discoveries has allowed the identification of CCDC6 as partner of genes other than RET in different tumor types. CCDC6 gene product has a proper role in sustaining the DNA damage checkpoints in response to DNA damage. The inactivation of CCDC6 secondary to chromosomal rearrangements or gene mutations could enhance tumor progression by impairing the apoptotic response upon the DNA damage exposure, contributing to the generation of radio- and chemoresistance. Preclinical studies indicate that the attenuation of CCDC6 in cancer, while conferring a resistance to cisplatinum, sensitizes the cancer cells to the small molecule inhibitors of Poly (ADP-ribose) polymerase (PARP1/2) with a synthetic lethal effect. Several CCDC6 mutations and gene rearrangements have been described so far in different types of cancer and CCDC6 may represent a possible predictive biomarker of tumor resistance to the conventional anticancer treatments. Nevertheless, the detection of a CCDC6 impairment in cancer patients may help to select, in future clinical trials, those patients who could benefit of PARP-inhibitors treatment alone or in combination with other treatments.

Chemokines as a Conductor of Bone Marrow Microenvironment in Chronic Myeloid Leukemia.

All blood lineage cells are generated from hematopoietic stem cells (HSCs), which reside in bone marrow after birth. HSCs self-renew, proliferate, and differentiate into mature progeny under the control of local microenvironments including hematopoietic niche, which can deliver regulatory signals in the form of bound or secreted molecules and from physical cues such as oxygen tension and shear stress. Among these mediators, accumulating evidence indicates the potential involvement of several chemokines, particularly CXCL12, in the interaction between HSCs and bone marrow microenvironments. Fusion between breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog (ABL)-1 gene gives rise to BCR-ABL protein with a constitutive tyrosine kinase activity and transforms HSCs and/or hematopoietic progenitor cells (HPCs) into disease-propagating leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). LSCs can self-renew, proliferate, and differentiate under the influence of the signals delivered by bone marrow microenvironments including niche, as HSCs can. Thus, the interaction with bone marrow microenvironments is indispensable for the initiation, maintenance, and progression of CML. Moreover, the crosstalk between LSCs and bone marrow microenvironments can contribute to some instances of therapeutic resistance. Furthermore, evidence is accumulating to indicate the important roles of bone marrow microenvironment-derived chemokines. Hence, we will herein discuss the roles of chemokines in CML with a focus on bone marrow microenvironments.

Abstract 5158: Anti-tumor activity of entrectinib, a highly potent pan-TRK, ROS1 and ALK inhibitor, in molecularly defined acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and comprises a heterogeneous group of diseases. A number of recurrent leukemogenic gene mutations or chromosomal rearrangements have been identified and clinically validated in AML. However, nearly 50% of AML patient samples lack any known AML driver mutations. Advances in molecular diagnostics have resulted in the identification of novel and actionable gene mutations or chromosomal rearrangements in these AML samples. The ETV6-NTRK3 fusion gene is one such rearrangement identified in samples from patients with AML. Fusion of ETV6 sequences to the tyrosine kinase domain of NTRK3 results in constitutive activation of the TRKC kinase and ETV6-NTRK3 expression has emerged as one of the key oncodrivers for leukemogenesis. Constitutive activation of TRK family tyrosine kinases has also been detected in a wide range solid tumor and hematologic malignancies, including lung, colorectal, salivary gland, sarcoma, thyroid, glioblastoma, melanoma, anaplastic large cell lymphoma (ALCL) and Philadelphia-like acute lymphoblastic leukemia. Entrectinib (RXDX-101) is an investigational, orally available, brain-penetrant, highly potent and selective kinase inhibitor with low nanomolar potency against TRKA/B/C, ROS1 and ALK kinase activities (encoded by NTRK1/2/3, ROS1 and ALK genes, respectively). In these studies, we have demonstrated sensitivity to entrectinib in AML cell lines with endogenous expression of the ETV6-NTRK3 fusion gene. Entrectinib treatment blocked cell proliferation and survival in vitro with sub-nanomolar EC50 values. Phosphorylation of the ETV6-TRKC fusion protein as well as phosphorylation of known TRKC downstream signaling effectors was inhibited by entrectinib treatment in a dose-dependent manner. Sensitivity to entrectinib was dependent on expression of the TRKC fusion protein. In xenograft models, entrectinib treatment at clinically relevant doses resulted in tumor regression, which was accompanied by elimination of residual cancer cells from the bone marrow. The clinical relevance of activated oncogenic tyrosine kinases resulting from chromosomal rearrangements has been validated by the efficacy of selective tyrosine kinase inhibitors. Our preclinical data demonstrate the potential of entrectinib as an effective treatment for patients with NTRK rearranged acute myeloid leukemias and provide rationale for the clinical development of entrectinib in molecularly defined hematologic malignancies. Entrectinib is currently the subject of an ongoing global Phase 2 basket study enrolling patients across multiple tumor histologies containing TRK, ROS1 or ALK fusions. Citation Format: Patrick Fagan, Maria Barrera, Colin Walsh, Danielle Murphy, Ian Silverman, Robert Shoemaker, Ge Wei, Zachary Hornby, Gary Li, Kristen M. Smith. Anti-tumor activity of entrectinib, a highly potent pan-TRK, ROS1 and ALK inhibitor, in molecularly defined acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5158. doi:10.1158/1538-7445.AM2017-5158

MPT0B002, a novel microtubule inhibitor, downregulates T315I mutant Bcr-Abl and induces apoptosis of imatinib-resistant chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy caused by the constitutive activation of Bcr-Abl tyrosine kinase. The Bcr-Abl inhibitor imatinib and other second-generation tyrosine kinase inhibitors such as dasatinib and nilotinib have remarkable efficacy in CML treatment. However, gene mutation-mediated drug resistance remains a critical problem. Among point mutations, the Bcr-Abl T315I mutation confers resistance to these Bcr-Abl inhibitors. Previously, we have synthesized the compound (1-methyl-1H-indol-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone (MPT0B002) as a novel microtubule inhibitor. In this study, we evaluated its effects on the proliferation, cell cycle, and apoptosis of K562 CML cells and BaF3 cells expressing either wild-type Bcr-Abl (BaF3/p210) or T315I-mutated Bcr-Abl (BaF3/T315I). MPT0B002 inhibited cell viability in a dose-dependent manner in these cells but did not affect the proliferation of human umbilical vein endothelial cells. It disrupted tubulin polymerization and arrested cell cycle at the G2/M phase. Treatment with MPT0B002 induced apoptosis, and this induction was associated with increased levels of cleaved caspase-3 and cleaved PARP. Furthermore, MPT0B002 can downregulate both Bcr-Abl and Bcr-Abl-T315I mRNA expressions and protein levels and the downstream signaling pathways. Taken together, our findings suggest that MPT0B002 may be considered a promising compound to downregulate not only wild type Bcr-Abl but also the T315I mutant to overcome Bcr-Abl-T315I mutation-mediated resistance in CML cells.

Therapeutic strategies and mechanisms of drug resistance in anaplastic lymphoma kinase (ALK)-rearranged lung cancer.

Anaplastic lymphoma kinase (ALK) gene encoding the receptor tyrosine kinase ALK is expressed as a fusion gene in a variety of carcinomas. The expression of ALK is nearly undetectable in adults, and its activation is normally regulated by its ligands, FAM150A/B. However, ALK gene rearrangements result in constitutive ALK fusion proteins expression via the active promoter of fusion partner genes. ALK fusion proteins dimerize in a ligand-independent manner and lead to the dysregulation of cell proliferation via abnormal constitutive activation of ALK tyrosine kinase. Many ALK tyrosine kinase inhibitors (TKIs) have been developed to date, three of which are currently in clinical use for the treatment of ALK-rearranged non-small cell lung cancer (NSCLC). ALK TKIs often achieve marked tumor regression in NSCLC patients with ALK rearrangements; however, ALK TKI-resistant tumors inevitably emerge within a few years in most cases. In this review, we summarize diverse ALK TKI resistance mechanisms identified in NSCLC with ALK rearrangements, and review potential therapeutic strategies to overcome ALK TKI resistance in these patients.

SC11.04 Rare Mutations in Lung Cancer

“Lung adenocarcinoma” is a genetically heterogenous disease entity, characterized by a wide spectrum of different mutations. Some of these mutations lead to constitutive activation of receptor tyrosine kinases, which can be inhibited by small molecules (tyrosine kinase inhibitors, TKIs). EGFR mutations (2004) and ALK rearrangement (2007) were among the first actionable driver mutations identified in lung adenocarcinomas. Today, several drugs are approved for the treatment of advanced lung adenocarcinomas with EGFR mutations or ALK/ROS1 rearrangement. Combined, these molecular subgroups make up at least 20% of all lung adenocarcinomas or more, depending on the poplulation. Further actionable driver mutations include the genes BRAF, HER2, MET, and RET. These genes are less frequently mutated than EGFR/ALK, nevertheless, rare drivers are clinically relevant because of the availability of targeted therapies approved for other indications in oncology (ALK-lung, HER2-breast, RET-thyroid, and BRAF-melanoma). The discussant will summarize current knowledge about rare driver mutations, with a strong clinical focus. HER2 insertion 20, present in about 1% of lung adenocarcinomas, was initially proposed by Cappuzzo et al as a potential indication for trastuzumab-based therapy.1 Prospective trials with HER2 targeting drugs are currently ongoing. BRAF V600E, present in about 3% of lung adenocarcinomas, was associated with high activity of combined therapy with dabrafenib and trametinib in a prospective phase II trial by Planchard et al.2 Crizotinib, recently approved by the FDA for the treatment of ROS1-NSCLC, is also active in tumors harboring MET exon 14 mutations as demonstrated by Drilon et al.3 Cabozantinib and vandetanib are active in tumors with RET rearrangement as shown by three recent phase II trials.4-6 Entrectinib showed preliminary activity in tumors harboring TRK rearrangement in an early basket Trial.7 These results will be discussed in detail, together with the results of international registries (EUHER2, EURAF, EUROS1 and GLORY).8 Moreover, current treatment recommendations for patients with advanced lung adenocarcinomas and rare driver mutations will be summarized. 1. Cappuzzo et al. N Engl J Med. 2006;354(24):2619-21. 2. Planchard et al. Lancet Oncol. 2016;17(7):984-93. 3. Drilon et al. J Clin Oncol 34, 2016 (suppl; abstr 108) 4. Drilon et al. Cancer Discov. 2013;3(6):630-5. 5. Seto et al. J Clin Oncol 2016;34(suppl; abstr 9012) 6. Lee et al. J Clin Oncol 2016;34(suppl; abstr 9013) 7. Drilon et al. AACR 2016 (abstract CT007) 8. Gautschi et al. WCLC 2016 (abstract 4325). NSCLC, Driver Mutations, Targeted Therapies

Ectopic Expression of Snail and Twist in Ph+ Leukemia Cells Upregulates CD44 Expression and Alters Their Differentiation Potential.

Philadelphia chromosome-positive (Ph+) leukemia is characterized by reciprocal translocation between chromosomes 9 and 22. The resultant BCR/ABL fusion protein displays constitutive tyrosine kinase activity, leading to the induction of aberrant proliferation and neoplastic transformation. The bone marrow (BM) microenvironment is tumor-promoting, and contributes to disease recurrence in Ph+ leukemia. Activity in the BM microenvironment is mediated by several cellular compartments, extracellular matrix, various soluble factors including transforming growth factor beta 1 (TGF-β1), and the hypoxic conditions in the BM niche. TGF-β1 is released during bone remodeling and plays a role in maintaining leukemic stem cells, as well as being implicated in the epithelial-mesenchymal transition (EMT) process in most solid tumors. Although EMT is largely implicated in epithelial tumors, recent findings argue for an EMT-like process in leukemia as well. The surface receptor CD44 is involved in cell adhesion, cell migration, and homing of normal and malignant hematopoietic stem cells. Elevation of CD44 expression is considered a marker for a worse prognosis in most hematological malignancies. We explored the functions of Snail and Twist1 in Ph+ leukemia. We showed that ectopic expression of Snail and, to a lesser extent, Twist1, upregulates CD44 expression that is β-catenin-dependent. Moreover, the presence of Snail or Twist1 partially blocked phorbol 12-myristate 13-acetate-induced megakaryocyte differentiation, while that of Twist significantly altered imatinib-induced erythroid differentiation. Thus EMT modulators affected proliferation, CD44 gene expression and differentiation ability of Ph+ leukemia cells.

The Polymorphisms on Chromosome 9p21 Play a Role in the Risk for Cardiovascular Events in Chronic Myeloid Leukemia Patients?

The polymorphisms on chromosome 9p21 play a role in the risk for cardiovascular events in chronic myeloid leukemia patients?Chronic myeloid leukemia (CML) is an onco-haematological disease due to the aberrant expression of an onco-protein with constitutive tyrosine kinase activity. The average age of onset is 55-60 years. The treatment with tyrosine kinase inhibitors (TKIs) drastically changed the outcome of patients affected by chronic myeloid leukemia, allowing long-term improvement in overall survival and deep molecular responses. TKI treatment is also associated to an increased risk of cardiovascular event. The etiopathogenesis of this effects is not clear possibily due to a damage to the endothelial cell, the results of the interaction of genetical predisposition, risk factors and life style habit. The 9p21 region is the strongest genomic marker for cardiovascular disease that has been identified in multiple genome-wide association study.We retrospectively studied 182 patients affected by CML and treated with different TKIs for expression of polymorphism rs1333040C>T and rs7865618A>G of chromosome 9p21 in order to define the role of genetic cardiovascular risk profile to better tailor individualized treatment strategy and identify patients who require strict monitoring of additional risk factors during treatment.Patients and methodsWe analysed 182 CML patients in chronic phase. All patients were treated with either first, second or third generation TKIs. Patients were compared to a control group including 171 subjects. Genomic DNA was isolated from peripheral blood and the rs1333040C>T and rs7865618A>G polymorphisms were assessed by PCR-RFLP using the BsmI and MspI restriction endonucleases, respectively.ResultsNinety-three out of 181 (51%) patients presented the C/T polymorphism, 82 (46%) presented T/T polymorphism and 6 (3%) patient presented wild type polymorphism C/C for rs1333040, test result was not evaluable in 1 patient.Eighty-five out of 181 (47%) patients presented the G/A polymorphism, 73 (40%) presented A/A polymorphism and 23 (13%) patient presented wild type polymorphism G/G for rs7875618 . Test result was not evaluable in 1 patient.The distribution of polymorphism C/C for rs1333040 and G/G for rs7875618 were statistically different compared to control group (p0.0004 and p0.0136 respectively) as showed in table 1. In a sub-analysis, including only 93 patients, a significantly higher incidence of cardiovascular events according to genotypes of SNP rs 1333040 was observed. In C/T group (54 patients) we retrospectively observed 3 cardiovascular events (5.55%) : 2 were transient ischemic attack (TIA) before the diagnosis of CML occurring in 2 female patients aged 69 and 73 respectively and a peripheral arterial obstructive disease (PAOD) in a female patient ,aged 74, with a baseline CAD score of 10, receiving nilotinib 800 mg/d as third line treatment.In T/T group (39 patients) we retrospectively observed 9 (23.1%) cardiovascular events at a median age of 60 years (range 44-82) and with a median estimated cardiovascular risk of 8 (range 0-20) Six patients developed a myocardial acute infarction (AMI) during treatment with TKIs, but 5 out of six had a previous history of AMI. One patient presented a TIA after history of previous of AMI. All patients with previous history of AMI were receving antiplatelets agents. One patient developed PAOD during first line treatment wih nilotinib and one developed an distal arteriopathy associated to an erectile dysfunction.The different incidence of cardiovascular events according to genotypes (T/T vs C/T ) was statistically significant by Fisher'sTest (p=0.0248).DiscussionThe role of 9p21 region , in particular the genes located in proximity of a noncoding RNA sequence named ANRIL, could be a useful guide for the prophylaxis of cardiovascular events during TKI therapy.Table 1 genotypes distribution between CML patients and controlsrs1333040 rs7865618Patients 82 93 6 23 85 7346% 51% 3% 13% 47% 40%Controls 62 84 25 8 77 8636% 49% 15% 5% 45% 50%p=0.0004 P=0.0136 DisclosuresBacigalupo:SANOFI: Speakers Bureau; PIERRE FABRE: Speakers Bureau.

SiRNA-cell-penetrating peptides complexes as a combinatorial therapy against chronic myeloid leukemia using BV173 cell line as model

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a single gene mutation, a reciprocal translocation that originates the Bcr-Abl gene with constitutive tyrosine kinase activity. As a monogenic disease, it is an optimum target for RNA silencing therapy. We developed a siRNA-based therapeutic approach in which the siRNA is delivered by pepM or pepR, two cell-penetrating peptides (CPPs) derived from the dengue virus capsid protein. These peptides have a dual role: siRNA delivery into cells and direct action as bioportides, i.e. intracellularly bioactive CPPs, targetting cancer-related signaling processes.Both pepM and pepR penetrate the positive Bcr-Abl+ Cell Line (BV173). Five in silico designed anti-Bcr-Abl siRNA were selected for in vitro analysis after thorough screening. The Bcr-Abl downregulation kinetics (48h to 168h) was followed by quantitative PCR. The bioportide action of the peptide vectors was evaluated by genome-wide microarray analysis and further validated by testing BV173 cell cycle and cell proliferation monitoring different genes involved in housekeeping/cell stress (RPL13A, HPRT1), cell proliferation (ki67), cell apoptosis (Caspase 3 and Caspase 9) and cell cycle steps (CDK2, CCDN2, CDKN1A). Assays with a commercial transfection agent were carried out for comparison purposes. Maximal Bcr-Abl gene knockdown was observed for one of the siRNA when delivered by pepM at 120h. Both pepM and pepR showed downregulation effects on proliferative CML-related signaling pathways having direct impact on BV173 cell cycle and proliferation, thus reinforcing the siRNA effect by acting as anticancer molecules.With this work we show the therapeutic potential of a CPP shuttle that combines intrinsic anticancer properties with the ability to deliver functional siRNA into CML cell models. By such combination, the pepM-siRNA conjugates lowered Bcr-Abl gene expression levels more extensively than conventional siRNA delivery technologies and perturbed leukemogenic cell homeostasis, hence revealing their potential as novel alternative scaffolds for CML therapy.

Roles of Interferon Regulatory Factors in Chronic Myeloid Leukemia.

The Interferon Regulatory Factor (IRF) family consists of multiple transcription factors involved in the regulation of a variety of biological processes. Originally identified as transcriptional regulators of the type I interferon system, IRFs play a pivotal role in adaptive immunity, cell growth, differentiation and tumorigenesis. Hence, understanding IRF biology has important implications in the host response to cancer development and progression. Many lines of evidence suggest that different IRFs are involved in the pathogenesis of Chronic Myeloid Leukemia (CML), a myeloproliferative disorder caused by the BCR-ABL oncoprotein. BCR-ABL displays constitutive tyrosine kinase activity that favors cell proliferation, inhibits apoptosis and allows cell survival even in the absence of proper adhesion to the extracellular matrix. Different BCR-ABL tyrosine kinase inhibitors are currently available for CML treatment. These drugs are able to generate eight year CML-specific overall survival rates >90%, only a minority of patients will achieve molecular responses compatible with drug discontinuation. Thus, there is an unmet need for additional therapeutic targets that may lead to the cure of most patients diagnosed with CML. A growing body of evidence has suggested a role for both IRF4 and IRF8 in the pathogenesis of CML. Furthermore, IRF1 is consistently deleted at one or both alleles in patients with leukemia and myelodysplasia. Finally, we have recently demonstrated that IRF5 is a target of BCR-ABL kinase activity and reduces CML cell proliferation. In this article, we provide an update on the current knowledge of the role of the IRFs in CML.

Reversine triggers mitotic catastrophe and apoptosis in K562 cells

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm of the hematopoietic stem cell characterized by presence of the oncoprotein BCR-ABL1, which have constitutive tyrosine kinase activity. BCR-ABL1 activation induces aurora kinase A (AURKA) and aurora kinase B (AURKB) expression, which are serine-threonine kinases that play an important function in chromosome alignment, segregation and cytokinesis during mitosis. Acquisition of resistance to tyrosine kinase inhibitors has emerged as a problem for CML patients and the identification of novel targets with an important contribution for CML phenotype is of interest. In the present study, we explored the cellular effects of reversine, an AURKA and AURKB inhibitor, in the BCR-ABL1+ K562 cells. Our results indicate that reversine reduces AURKA and AURKB expression, leads to reduction of cell viability and increased apoptosis in a dose- and time-dependent manner, as well as, induces mitotic catastrophe in K562 cells. Our preclinical study establishes that reversine presents an effective antileukemia activity against K562 cells and provide new insights on anticancer opportunities for CML.

Effects of the multikinase inhibitors Sorafenib and Regorafenib in PTEN deficient neoplasias

The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) axis is frequently dysregulated in cancer due to mutations in different nodes of the pathway or constitutive activation of receptor tyrosine kinases. Multikinase inhibitors as sorafenib and regorafenib represent a therapeutic approach for the treatment of these types of tumours. In the present study, we have evaluated the anti-tumoural effects of Sorafenib and Regorafenib on endometrial, prostate and thyroid neoplasias. Both inhibitors reduced cell viability in vitro and lead to a disruption of the PI3K/AKT/mTOR pathway. In vivo, we have demonstrated that Sorafenib and Regorafenib reduce thyroid hyperplasias induced by the loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), although none of the treatments eliminated the disease. Altogether, we present the first study that correlates the response to multikinase inhibitors with a specific mutation. Moreover, this is the first report characterising the response to Regorafenib in thyroid, prostate and endometrial neoplasias.

ROS1 fusions in cancer: a review.

The ROS1 gene belongs to the sevenless subfamily of tyrosine kinase insulin receptor genes. A literature review identified a ROS1 fusion in 2.54% of the patients with lung adenocarcinoma and even higher frequencies in spitzoid neoplasms and inflammatory myofibroblastic tumors. At present, 26 genes were found to fuse with ROS1, some of them already known to fuse with RET and ALK. All the fusion proteins retain the ROS1 kinase domain, but rarely its transmembrane domain. Most of the partners have dimerization domains that are retained in the fusion, presumably leading to constitutive ROS1 tyrosine kinase activation. Some partners have transmembrane domains that are retained or not in the chimeric proteins. Therefore, different ROS1 fusions have distinct subcellular localization, suggesting that they may activate different substrates in vivo.

New Insights into the Molecular Resistance Mechanisms of Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the causative oncoprotein BCR-ABL1 (Breakpoint-cluster region/Abelson kinase), which is a fusion protein with constitutive tyrosine kinase activity. The first tyrosine kinase inhibitor (TKI), imatinib, revolutionized the treatment of CML. Despite the spectacular effects of imatinib, primary and acquired resistance as well as intolerance to imatinib still exist. The mechanisms of BCR-ABL1 point mutations, amplification of the BCR-ABL1 gene and increased expression of efflux drug transporters, which play important roles in resistance, have been extensively described. Consequently, second- and third-generation TKIs have been explored to overcome resistance. However, some CML patients are refractory to all available TKIs. In addition, most patients relapse after discontinuing TKI therapy, due to the existence of CML stem cells, which have been demonstrated to be primarily resistant to TKIs. Thus, TKIs alone are not sufficient to cure CML, and it is necessary to further investigate the molecular resistance mechanisms in both the bulk and stem cells of CML to identify new targets to overcome resistance and eradicate the residual CML stem cells. This article reviews new insights into the various molecular resistance mechanisms of CML and discusses treatment strategies based on the targets that have recently been found to play an important role in the molecular mechanisms of resistance.

Inhibition of protein kinase CK2 by CX-5011 counteracts imatinib-resistance preventing rpS6 phosphorylation in chronic myeloid leukaemia cells: new combined therapeutic strategies.

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder promoted by the constitutive tyrosine kinase activity of Bcr-Abl oncoprotein. Although treatment with the Bcr-Abl-inhibitor imatinib represents the first-line therapy against CML, almost 20-30% of patients develop chemotherapeutic resistance and require alternative therapy. Here we show that a strong hyper-phosphorylation/activation of ERK1/2, Akt Ser473, and 40S ribosomal protein S6 (rpS6) is detectable in imatinib-resistant KCL22 and K562 CML cells as compared to the -sensitive cell variants. In imatinib-resistant CML cells, high concentration of imatinib is required to strongly inhibit Bcr-Abl, ERK1/2 and Akt Ser473 phosphorylation, but under these conditions the phosphorylation of rpS6, a common downstream effector of MEK/ERK1/2 and PI3K/Akt/mTOR pathways is only slightly reduced. By contrast, down-regulation of the protein kinase CK2 by the inhibitor CX-5011 or by silencing the CK2 subunits does not affect the activation state of MEK/ERK1/2 or PI3K/Akt/mTOR signalling, but causes a drop in rpS6 phosphorylation in parallel with reduced protein synthesis. CK2-inhibition by CX-5011 induces cell death by apoptosis and acts synergistically with imatinib or the MEK-inhibitor U0126 in reducing the viability of imatinib-resistant CML cells. The ternary mixture containing CX-5011, imatinib and U0126 represents the most effective synergistic combination to counteract CML cell viability.These results disclose a novel CK2-mediated mechanism of acquired imatinib-resistance resulting in hyper-phosphorylation of rpS6. We suggest that co-targeting CK2 and MEK protein kinases is a promising strategy to restore responsiveness of resistant CML cells to imatinib.

Induction of Chronic Myeloid Leukemia in Mice.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder derived from a hematopoietic stem cell (HSC), harboring Philadelphia chromosome (Ph chromosome). Formation of the Ph chromosome is caused by a reciprocal translocation between the chromosomes 9 and 22 t(9;22)(q34;q11), resulting in a fusion protein known as BCR-ABL which has constitutive tyrosine kinase activity and promotes the proliferation of leukemia cells via multiple mechanisms. Studies on CML have led to the identification of the first cancer-associated chromosomal abnormality and the subsequent development of tyrosine kinase inhibitors (TKIs) that inhibit BCR-ABL kinase activity in CML. It has become clear that leukemia stem cells (LSCs) in CML are insensitive to inhibition by TKIs, and eradication of LSCs appears to be difficult. Therefore, some of the major issues in current CML therapy are to understand the biology of LSCs and to investigate why LSCs are insensitive to TKIs for developing curative therapeutic strategies. In this regard, application of mouse models recapitulating human CML disease will be critical. In this chapter, we describe methods for induction of CML in mice with BCR-ABL.

Critical Role of SHP2 in the Initiation and Maintenance of Myeloproliferative Neoplasms Evoked By JAK2V617F

The JAK2V617F mutation has been found in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of the JAK2 tyrosine kinase and its downstream signaling pathways. JAK2 inhibitor therapy can reduce splenomegaly and constitutional symptoms but is not sufficient to cure or produce complete remission in patients with MPNs. Identification of critical signaling pathways/molecules required for the initiation and maintenance of MPNs will facilitate the development of more effective targeted therapies.To determine the role of JAK2V617F mutation in MPNs, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mice hematopoietic compartments is sufficient to induce a PV-like MPN, whereas homozygous Jak2V617F expression accelerates the development of myelofibrosis. We have found that SHP2, a protein tyrosine phosphatase that positively regulates hematopoietic signaling, is constitutively phosphorylated in mouse and human hematopoietic cells expressing JAK2V617F. However, the contribution of SHP2 in the pathogenesis of MPNs induced by JAK2V617F remains elusive. Here, we sought to determine the role of SHP2 in JAK2V617F-evoked MPNs using conditional SHP2 knockout (SHP2 floxed) and Jak2V617F knock-in mice and MxCre line.Whereas expression of heterozygous Jak2V617F induced a PV-like MPN characterized by increase in red blood cells (RBC), white blood cells (WBC), neutrophils and platelets in the peripheral blood and splenomegaly, deletion of SHP2 normalized the blood parameters and spleen size in Jak2V617F knock-in mice. Flow cytometric analysis showed that deletion of SHP2 inhibited the expansion of erythroid, megakaryocytic, and granulocytic precursors in the bone marrow (BM) and spleens of Jak2V617F mice. Deletion of SHP2 also inhibited the expansion of hematopoietic stem cells and megakaryocyte-erythroid progenitors in the BM and spleens of Jak2V617F knock-in mice. Furthermore, deletion of SHP2 markedly inhibited the erythropoietin (Epo)-independent CFU-E colonies in the BM and spleens of Jak2V617F mice.In order to determine whether the effects of SHP2 deletion in Jak2V617F mice were cell autonomous, BM cells from uninduced control, MxCre;V617F/+ and MxCre;V617F/+;SHP2fl/fl mice were transplanted into lethally irradiated syngeneic recipient mice. Four weeks after transplantation, mice were injected with pI-pC to induce deletion of SHP2 and expression of Jak2V617F simultaneously. Transplanted animals receiving MxCre;V617F/+ BM developed a PV-like disease within 6 weeks after pI-pC induction. However, recipients of MxCre;V617F/+;SHP2fl/fl BM failed to induce the PV disease due to deletion of SHP2. Together, these data suggest that SHP2 is required for the initiation of PV-like MPN mediated by Jak2V617F.To determine if SHP2 is required for the maintenance of MPN evoked by Jak2V617F, uninduced wild type or MxCre;SHP2fl/fl mice BM cells were transduced with retroviruses expressing Jak2V617F and transplanted into lethally irradiated syngeneic recipient animals. Five weeks after transplantation, we assessed the peripheral blood counts to confirm the development of MPN disease. Transplanted animals receiving Jak2V617F-transduced wild type or MxCre;SHP2fl/fl mice BM were then divided into two groups- one group was injected with pI-pC (to induce deletion of SHP2 after establishment of the MPN disease); another group (control group) was injected with saline. We observed that deletion of SHP2 by pI-pC induction significantly inhibited the RBC, hematocrit, WBC and platelet counts in the peripheral blood and reduced the spleen size in transplanted animals expressing Jak2V617F. Moreover, deletion of SHP2 markedly inhibited the Epo-independent erythroid colonies in the BM and spleens of transplanted animals expressing Jak2V617F. Thus, SHP2 is not only required for the initiation of MPN but also required for the maintenance of MPN mediated by Jak2V617F. We have also observed that deletion of SHP2 blocks the development of myelofibrosis in Jak2V617F knock-in mice. In conclusion, our results suggest that SHP2 plays a critical role in the initiation and maintenance of MPNs evoked by Jak2V617F. DisclosuresNo relevant conflicts of interest to declare.

Refining Triage to Polycythaemia Vera or Primary Myelofibrosis Based on Targeted Molecular Profiling By Clinical Next Generation Sequencing

Background: Primary myelofibrosis (PMF) and polycythemia vera (PV) are myeloproliferative neoplasms (MPN) that can both share a similar bone marrow morphology with panmyelosis and fibrosis, posing a diagnostic challenge, particularly when the differential is between cellular phase of PMF and PV, or fibrotic PMF and post-PV myelofibrosis. Despite advances in genomic analysis, limited information is known regarding their differences in genetic profile/signature. It has been well known that constitutive tyrosine kinase activation due to JAK2 V617F mutation is seen in both PV and PMF. MPL and CALR mutations do segregate with PMF but may not be found in all cases. Accordingly, we analyzed next generation sequencing (NGS) data to look for potential biomarkers that may further aid in distinguishing these two entities.Design: The IRB approved study intended to recruit patients with diagnosis of PMF and PV who have myeloid gene mutation profiles available. Clinical information and molecular data from both a CLIA certified reference laboratory and our institution from May 2011 to June 2015 were retrieved. Cases with other myeloid neoplasms were excluded. The gene mutation profiles by Next Generation sequencing (NGS) and conventional karyotyping were acquired and compared. Clinicopathologic features including disease progression, degree of fibrosis in bone marrow, percentage of blasts, bone marrow cellularity, and circulating blood count (CBC) are correlated. Student t-test was used for numerical variables and Chi square (x2) test was used for categorical variables.Results: Of the 62 patients qualified in the study, 36 patients were diagnosed with PMF (Age 68.5 ± 12.2, M:F ratio of 1:1) and 26 patients with PV (Age 66.5 ± 11.9, M:F ratio of 1.6). The majority of patients (34/36 PMF and 26/26 PV) showed persistent disease with only two PMF patients progressing to acute myeloid leukemia (AML). In accordance with prior reports, JAK2 V617F mutation was more prevalent in PV (23/26, 88%) than in PMF (17/36, 47%)(p<0.05), while MPL mutation was found in PMF (5/36, 14%) but not in PV (0/26) (p<0.001). Overall, PMF patients tended to have more non JAK2 mutations (mean = 1.6 ± 1) than PV patients (mean= 0.54 ± 0.65) (p = 0.005), even though the PV patients tended to have a longer history of disease. Interestingly, ASXL1 mutations (mainly frame-shift, reportedly pathologic) appear to be more prevalent in PMF (28%) than in PV (8%) patients (p = 0.058). SRSF2 mutations were found in 14% of PMF patients but absent in all 26 PV patients (p=0.068). Mutations in a subset of other analyzed genes (TET2, EZH2, IDH2, and CUX1) were also more frequent in PMF than in PV patients (25% vs 15%, 8% vs 0%, 8% vs 0%, and 6% vs 0%, respectively), but not statistically significant due to limited number of cases. The highest number of mutations (n=4) was in a case of PMF that progressed to AML, suggesting a 'dosage' effect of driver mutations on outcomes similar to that described in MDS. The other patient that progressed from PMF to AML harbored JAK2, ASXL1, SRSF2 mutations along with del(20q). ASXL1 mutation was associated with del(20q) in 4/62 cases, all of which were PMF patients including the case that has progressed to AML. JAK2 mutation was associated with del(20q) in 7 out of the 62 cases, 6 (86%) of which were PMF patients. No gene mutations were uniquely associated with degree of fibrosis, blast count, cellularity, white blood cell counts, hemoglobin, or platelet counts.Conclusion: Our results indicate that PMF patients tend to have more non JAK2 mutations (e.g., ASXL1, SRSF2) than PV. Furthermore, the mutations, including JAK2 mutations, are more likely to be associated with del(20q) in PMF patients. Our findings provide insight into the genetic landscape of PMF and PV and offer potential biomarkers that may be helpful to distinguish between these entities, thus benefiting patient stratification for clinical practice. DisclosuresLancet:Seattle Genetics: Consultancy; Pfizer: Research Funding; Boehringer-Ingelheim: Consultancy; Kalo-Bios: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Research Funding. Komrokji:Celgene: Consultancy, Research Funding; Incite: Consultancy; Novartis: Speakers Bureau; GSK: Research Funding.

Abstract LB-B19: BKM-120: a phosphatidyl-inositol-3 kinase inhibitor with anti-migratory properties in glioblastoma

Abstract Glioblastoma (GBM) is the most common glial brain tumor and is also one of the most lethal human cancers: patients have a median survival of 15 months with a five-year survival rate of only 3%. The current standard-of-care has remained the same for the last decade and consists of maximal safe surgical resection followed by radio- and chemotherapy. GBM cells are highly infiltrative, leading to invasion of normal brain tissue by tumor cells. These invasive tumor cells render GBM a surgically incurable disease and tumor recurrence is almost inevitable, with 90% of patients developing new lesions within 2-3 cm of the original site or at distant sites in the brain. Moreover, invasion may increase further during anti-angiogenic therapy with bevacizumab and no anti-invasive approaches are yet available clinically. The phosphatidyl-inositol-3 kinase (PI3K) pathway is frequently deregulated in cancer and is activated in the majority of GBM cases due to constitutive receptor tyrosine kinase activation as well as inactivating mutations/deletions of PTEN (33%) or activating PI3K mutations (17%). In addition PI3K plays a role in cell migration in some cell types and a number of small molecule PI3K inhibitors are under investigation in oncology clinical trials. BKM-120 (Buparlisib), is a CNS-penetrant selective pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for several types of solid tumor, including GBM. We initially observed that BKM-120 is a potent anti-invasive molecule in GBM cell lines and patient-derived glioma stem-like cells in vitro. The anti-migratory effects of BKM-120 were clearly distinguishable from cytostatic and cytotoxic effects that occurred at higher drug concentrations and after a longer duration of drug exposure. The blockade of migration was reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenograft model. Mechanistically we found that GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. However, BKM-120 substantially reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition may contribute to the anti-invasive effects of BKM-120. Overall, our data suggest that the CNS-penetrant PI3K inhibitor BKM-120 may be a useful anti-migratory drug for the treatment of highly invasive tumors such as glioblastoma. Citation Format: Maria Carmela Speranza, Michal O. Nowicki, Choi-Fong Cho, E. Antonio Chiocca, Sean E. Lawler. BKM-120: a phosphatidyl-inositol-3 kinase inhibitor with anti-migratory properties in glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B19.

Association of promoter polymorphisms of Fas -FasL genes with development of Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a monoclonal myeloproliferative disorder of hematopoietic stem cells (HSCs), characterized by reciprocal translocation, leading to the formation of BCR-ABL oncogene with constitutive tyrosine kinase (TK) activity. This oncogene is known to deregulate different downstream pathways which ultimately lead to cell proliferation, defective DNA repair, and inhibition of apoptosis. Fas (Fas cell surface death receptor) is a member of tumor necrosis factor (TNF) superfamily which interacts with its ligand, FasL, to initiate apoptosis. Promoter polymorphisms in Fas-FasL genes are known to influence the apoptotic signaling. Hence, the present study has been aimed to find out the association of the promoter polymorphisms in Fas and FasL genes with the development and progression of CML. Blood samples from 772 subjects (386 controls and 386 cases) were collected and genotyped for Fas-FasL gene polymorphisms through PCR-RFLP method. The association between SNPs and clinical outcome was analyzed using statistical softwares like SPSS version 20, SNPSTATs, and Haploview 2.1. The study revealed a significant association of Fas -670 G>A and FasL -844 T>C polymorphisms with the development of CML while Fas -670 AG was associated with accelerated phase. Combined risk analysis by taking the risk genotypes in cases and controls revealed a significant increase in CML risk with increase in number of risk genotypes (one risk genotype-OR 1.99 (1.44-2.76), p < 0.0001; two risk genotypes-OR 3.33 (1.91-5.81), p < 0.0001). Kaplan-Meier survival analysis of Fas -670 A>G and FasL -844 T>C showed reduced event-free survival in patients carrying the variant genotypes, Fas -670 GG, 32.363±6.33, and FasL -844 CC, 33.489±5.83, respectively. Our findings revealed a significant association of Fas -670 GG, FasL -844 TC, and CC genotypes with increased risk of CML.