Detection of BCR-ABL kinase domain mutations in CD34+ cells from newly diagnosed chronic phase CML patients and their association with imatinib resistance

BCR-ABL kinase domain (KD) mutations, the most common cause of imatinib resistance, are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CP-CML) patients. Recent studies indicate pre-existing mutations (PEMs) can be detected in a higher percentage of CML patients using CD34+ stem/progenitor cells, and these mutations may correlate with imatinib resistance. We investigated KD mutations in CD34+ stem cells from 100 CP-CML patients by multiplex ASO-PCR and sequencing ASO-PCR products at the time of diagnosis. PEMs were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8-48), all patients with PEMs exhibited imatinib resistance. Of 68 patients without PEMs, 24 developed imatinib resistance. Mutations were detected in 21 of these patients by ASO-PCR and KD sequencing. All 32 patients with PEMs had the same mutations. In imatinib-resistant patients without PEMs, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients without T315I and Y253F mutations responded to imatinib dose escalation. In conclusion, BCR-ABL PEMs can be detected in a substantial number of CP-CML patients when investigated using CD34+ stem/progenitor cells. These mutations are associated with imatinib resistance, and mutation testing using CD34+ cells may facilitate improved, patient-tailored treatment

A rare case of melanosis of the hard palate mucosa in a patient with chronic myeloid leukemia

Imatinib Mesylate, also known as Gleevec or ST1-571, is a tyrosine-kinase inhibitor used as the gold standard medication for the chronic myeloid leukemia (CML); Imatinib has indeed deeply revolutionized the CML therapy allowing most patients to have a good quality of life. Despite its beneficial effects, Imatinib has significant side effects such as mucosal pigmentation. A 72-year-old female having an Imatinib induced mucosal pigmentation is presented: she has been treated with Imatinib since 2003 and only in 2014 discovered, during a routine dental visit, having a pigmented lesion on her hard palate mucosa. Histopathologically, the lesion shows the deposition of fine dark brown spherical bodies within the lamina propria and cloaked in between the collagen fibers. There was no sign of inflammation, hyperplasia, or hemorrhage in the tissu

Management of imatinib-resistant CML patients

Imatinib has had marked impact on outcomes in chronic myelogenous leukemia (CML) patients for all stages of the disease and is endorsed by international treatment guidelines as the first line option. Although imatinib is highly effective and well tolerated, the development of resistance represents a clinical challenge. Since the most frequently identified mechanism of acquired imatinib resistance is bcr-abl kinase domain point mutations, periodic hematologic, cytogenetic, and molecular monitoring is critical throughout imatinib therapy. Once cytogenetic remission is achieved, residual disease can be monitored by bcr-abl transcript levels as assayed by reverse transcription polymerase chain reaction (RT-PCR). Detection of bcr-abl mutants prior to and during imatinib therapy can aid in risk stratification as well as in determining therapeutic strategies. Thus, mutation screening is indicated in patients lacking or losing hematologic response. Moreover, search for mutations should also be performed when a 3-log reduction of bcr-abl transcripts is not achieved or there is a reproducible increase of transcript levels. In patients harboring mutations which confer imatinib resistance, novel second line tyrosine kinase inhibitors have demonstrated encouraging efficacy with low toxicity. Only the T315I bcr-abl mutant has proved totally resistant to all clinically available bcr-abl inhibitors. Strategies to further increase the rates of complete molecular remissions represent the next frontier in the targeted therapy of CML patients

Safe discontinuation of nilotinib in a patient with chronic myeloid leukemia: a case report

Case presentation. We report the case of a 64-year-old Caucasian man diagnosed with chronic-phase chronic myeloid leukemia in April 2005. After 4 years of treatment with imatinib, he became intolerant to the drug and was switched to nilotinib. Two years later, he decided to stop nilotinib. Undetectable molecular response persisted for 30 months after discontinuation of the drug. Introduction. Although there is a considerable amount of data in the literature on safe discontinuation of first-generation tyrosine kinase inhibitor therapy in patients with chronic myeloid leukemia, little is known about discontinuation of second-generation tyrosine kinase inhibitor therapy. Most previous studies have been focused on dasatinib, and the few cases of nilotinib withdrawal that have been reported had a median follow-up of 12 months. To the best of our knowledge, the present report is the first to describe nilotinib withdrawal with 30 months of follow-up. Conclusion: Our present case suggests that nilotinib withdrawal is safe for patients with chronic myeloid leukemia who achieve a stable undetectable molecular response. Our patient was homozygous for killer immunoglobulin-like receptor haplotype A, previously reported to be a promising immunogenetic marker for undetectable molecular response. We recommend additional studies to investigate patient immunogenetic profiles and their potential role in complete response to therap

Nilotinib, an approved leukemia drug, inhibits smoothened signaling in Hedgehog-dependent medulloblastoma.

Dysregulation of the seven-transmembrane (7TM) receptor Smoothened (SMO) and other components of the Hedgehog (Hh) signaling pathway contributes to the development of cancers including basal cell carcinoma (BCC) and medulloblastoma (MB). However, SMO-specific antagonists produced mixed results in clinical trials, marked by limited efficacy and high rate of acquired resistance in tumors. Here we discovered that Nilotinib, an approved inhibitor of several kinases, possesses an anti-Hh activity, at clinically achievable concentrations, due to direct binding to SMO and inhibition of SMO signaling. Nilotinib was more efficacious than the SMO-specific antagonist Vismodegib in inhibiting growth of two Hh-dependent MB cell lines. It also reduced tumor growth in subcutaneous MB mouse xenograft model. These results indicate that in addition to its known activity against several tyrosine-kinase-mediated proliferative pathways, Nilotinib is a direct inhibitor of the Hh pathway. The newly discovered extension of Nilotinib's target profile holds promise for the treatment of Hh-dependent cancers

The effects of time valuation in cancer optimal therapies: a study of chronic myeloid leukemia

Background The mathematical design of optimal therapies to fight cancer is an important research field in today’s Biomathematics and Biomedicine given its relevance to formulate patient-specific treatments. Until now, however, cancer optimal therapies have considered that malignancy exclusively depends on the drug concentration and the number of cancer cells, ignoring that the faster the cancer grows the worse the cancer is, and that early drug doses are more prejudicial. Here, we analyze how optimal therapies are affected when the time evolution of treated cancer is envisaged as an additional element determining malignancy, analyzing in detail the implications for imatinib-treated Chronic Myeloid Leukemia. Methods Taking as reference a mathematical model describing Chronic Myeloid Leukemia dynamics, we design an optimal therapy problem by modifying the usual malignancy objective function, unaware of any temporal dimension of cancer malignance. In particular, we introduce a time valuation factor capturing the increase of malignancy associated to the quick development of the disease and the persistent negative effects of initial drug doses. After assigning values to the parameters involved, we solve and simulate the model with and without the new time valuation factor, comparing the results for the drug doses and the evolution of the disease. Results Our computational simulations unequivocally show that the consideration of a time valuation factor capturing the higher malignancy associated with early growth of cancer and drug administration allows more efficient therapies to be designed. More specifically, when this time valuation factor is incorporated into the objective function, the optimal drug doses are lower, and do not involve medically relevant increases in the number of cancer cells or in the disease duration. Conclusions In the light of our simulations and as biomedical evidence strongly suggests, the existence of a time valuation factor affecting malignancy in treated cancer cannot be ignored when designing cancer optimal therapies. Indeed, the consideration of a time valuation factor modulating malignancy results in significant gains of efficiency in the optimal therapy with relevant implications from the biomedical perspective, specially when designing patient-specific treatments.This work was supported by projects MTM2014-56022-C2-2-P and MTM2017-85476-C2-1-P of the Spanish Office of Innovation and Competitiveness and European FEDER Funds, and by projects of the Castile and León Autonomous Government: VA041P17 (with European FEDER Funds), VA138G18 and VA148G18

Integrating microfluidics and biosensing on a single flexible acoustic device using hybrid modes

Integration of microfluidics and biosensing functionalities on a single device holds promise in continuous health monitoring and disease diagnosis for point-of-care applications. However, the required functions of fluid handling and biomolecular sensing usually arise from different actuation mechanisms. In this work, we demonstrate that a single acoustofluidic device, based on a flexible thin film platform, is able to generate hybrid waves modes, which can be used for fluidic actuation (Lamb waves) and biosensing (thickness shear waves). On this integrated platform, we show multiple and sequential functions of mixing, transport and disposal of liquid volumes using Lamb waves, whilst the thickness bulk shear waves allow us to sense the chemotherapeutic Imatinib, using an aptamer-based strategy, as would be required for therapy monitoring. Upon binding, the conformation of the aptamer results in a change in coupled mass, which has been detected. This platform architecture has the potential to generate a wide range of simple sample-to-answer biosensing acoustofluidic devices

Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1-dependent ABL1 activation in endothelial cells.

To enable new blood vessel growth, endothelial cells (ECs) express neuropilin 1 (NRP1), and NRP1 associates with the receptor tyrosine kinase VEGFR2 after binding the vascular endothelial growth factor A (VEGF) to enhance arteriogenesis. We report that NRP1 contributes to angiogenesis through a novel mechanism. In human and mouse ECs, the integrin ligand fibronectin (FN) stimulated actin remodeling and phosphorylation of the focal adhesion component paxillin (PXN) in a VEGF/VEGFR2-independent but NRP1-dependent manner. NRP1 formed a complex with ABL1 that was responsible for FN-dependent PXN activation and actin remodeling. This complex promoted EC motility in vitro and during angiogenesis on FN substrates in vivo. Accordingly, both physiological and pathological angiogenesis in the retina were inhibited by treatment with Imatinib, a small molecule inhibitor of ABL1 which is widely used to prevent the proliferation of tumor cells that express BCR-ABL fusion proteins. The finding that NRP1 regulates angiogenesis in a VEGF- and VEGFR2-independent fashion via ABL1 suggests that ABL1 inhibition provides a novel opportunity for anti-angiogenic therapy to complement VEGF or VEGFR2 blockade in eye disease or solid tumor growth

Protein kinase Cδ and c-Abl kinase are required for transforming growth factor β induction of endothelial-mesenchymal transition in vitro.

OBJECTIVE: The origin of the mesenchymal cells responsible for the intimal fibrosis in systemic sclerosis (SSc) has not been fully identified. The present study was undertaken to investigate whether subendothelial mesenchymal cells may emerge through transdifferentiation of endothelial cells (ECs) into myofibroblasts via endothelial-mesenchymal transition (EndoMT) in vitro and to explore the signaling pathways involved in this process. METHODS: Primary mouse pulmonary ECs isolated by immunomagnetic methods with sequential anti-CD34 and anti-CD102 antibody selection were cultured in monolayers. Cell morphology and diacetylated low-density lipoprotein uptake assays confirmed their EC characteristics. The induction of EndoMT was assessed by determination of α-smooth muscle actin (α-SMA), type I collagen, and VE-cadherin expression, and the expression of the transcriptional repressor Snail-1 was analyzed. The signaling pathways involved were examined using small-molecule kinase inhibitors and RNA interference. RESULTS: Transforming growth factor β1 (TGFβ1) induced α-SMA and type I collagen expression and inhibited VE-cadherin. These effects were mediated by a marked increase in Snail-1 expression and were abolished by treatment with either the c-Abl tyrosine kinase inhibitor imatinib mesylate or the protein kinase Cδ (PKCδ) inhibitor rottlerin. The inhibitory effects of imatinib mesylate and rottlerin were mediated by inhibition of phosphorylation of glycogen synthase kinase 3β at residue Ser(9). These observations were confirmed in experiments using small interfering RNA specific for c-Abl and PKCδ. CONCLUSION: These results indicate that c-Abl and PKCδ are crucial for TGFβ-induced EndoMT and that imatinib mesylate and rottlerin or similar kinase inhibitor molecules may be effective therapeutic agents for SSc and other fibroproliferative vasculopathies in which EndoMT plays a pathogenetic role