[Show abstract][Hide abstract] ABSTRACT: Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1-10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.
PLoS ONE 06/2015; 10(6):e0130360. DOI:10.1371/journal.pone.0130360 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.
PLoS ONE 07/2014; 9(7):e101848. DOI:10.1371/journal.pone.0101848 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon Regulatory Factor 5 modulates the expression of genes controlling cell growth and apoptosis. Previous findings have suggested a lack of IRF5 transcripts in both acute and chronic leukemias. However, to date, IRF5 expression and function have not been investigated in Chronic Myeloid Leukemia. We report that IRF5 is expressed in CML cells where it interacts with the BCR-ABL kinase that modulates its expression and induces its tyrosine phosphorylation. Tyrosine phosphorylated IRF5 displayed reduced transcriptional activity that was partially restored by Imatinib Mesylate. Interestingly, a mutant devoid of a BCR-ABL consensus site (IRF5(Y104F)) still presented significant tyrosine phosphorylation. This finding suggests that the oncoprotein phosphorylates additional tyrosine residues or induces downstream signaling pathways leading to further IRF5 phosphorylation. We also found that ectopic expression of IRF5 decreases the proliferation of CML cell lines by slowing their S-G2 transition, increasing the inhibition of BCR-ABL signaling and enhancing the lethality effect observed after treatment with IM, alpha-2-IFN and a DNA-damaging agent. Furthermore, IRF5 overexpression successfully reduced the clonogenic ability of CML CD34-positive progenitors before and after exposure to the above-indicated cytotoxic stimuli. Our data identify IRF5 as a downstream target of the BCR-ABL kinase, suggesting that its biological inactivation contributes to leukemic transformation.
[Show abstract][Hide abstract] ABSTRACT: Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR-ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR-ABL protein stability but are critical for preserving catalytic activity. Indeed, only a "conservative" I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR-ABL(I360T) revealed differences in both helix αC dynamics and protein-correlated motions, consistent with a modified ATP-binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR-ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR-ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.-Buffa, P., Romano, C., Pandini. A., Massimino, M., Tirrò, E., Di Raimondo, F., Manzella, L., Fraternali. F., Vigneri, P. G. BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein.
The FASEB Journal 12/2013; 28(3). DOI:10.1096/fj.13-236992 · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer incidence and mortality are higher among diabetic patients. This review examines the mechanisms, both general and site-specific, for this increase. Hyperglycemia and hyperinsulinemia, which are the major abnormalities that characterize diabetes, can promote cancer via both independent and synergic mechanisms. Insulin is both a metabolic hormone and a growth factor that promotes cell proliferation. When insulin levels are increased due to either insulin resistance or insulin treatment, their mitogenic effect is more marked in malignant cells that frequently overexpress the insulin receptor and, more specifically, its A isoform that has predominant mitogenic activity. Hyperglycemia provides energy for malignant cell proliferation and, via the peculiar energy utilization of cancer cells, favors cancer growth and neoangiogenesis. Additionally, diabetes-associated obesity has cancer-promoting effects due to mechanisms that are specific to excess fat cells (such as increased peripheral estrogens, increased pro-mitogen cytokines and growth factors). Also fat-associated chronic inflammation can favor cancer via the cell damage caused by reactive oxygen species (ROS) and via the production of inflammatory cytokines and transcription factors that stimulate cancer growth and invasiveness. Finally, the multiple drugs involved in the treatment of diabetes can also play a role. Diabetes-associated comorbidities, tissue-specific inflammation, and organ-specific dysfunctions can explain why the risk of cancer can differ by tissue type among diabetic patients. The increased risk of cancer-related mortality is moderate among individual patients with diabetes (RR = 1.25), but the pandemic nature of the disease means that a considerable number of lives could be spared through a better understanding of the factors associating diabetes and cancer.
[Show abstract][Hide abstract] ABSTRACT: As a group of more than 100 experts in chronic myeloid leukemia (CML), we draw attention to the high prices of cancer drugs, with the particular focus on the prices of approved tyrosine kinase inhibitors for the treatment of CML. This editorial addresses the multiple factors involved in cancer drug pricing and their impact on individual patients and health care policies, and argues for the need to (1) lower the prices of cancer drugs to allow more patients to afford them and (2) maintain sound long-term health care policies.
[Show abstract][Hide abstract] ABSTRACT: Context. Poorly differentiated thyroid carcinomas are refractory to common anti-cancer therapies, and novel inhibitors are being tested in these deadly malignancies. The EGF receptor (EGFR) tyrosine kinase represents an attractive target for treatment because it is up-regulated in thyroid cancer and plays a role in cancer progression. However, EGFR inhibitors have provided poor results in thyroid carcinomas. Objective. We evaluated the possible mechanism underlying resistance of thyroid cancer cells to EGFR inhibitors. Design. We tested the effect of the EGFR tyrosine kinase (TK) inhibitor Gefitinib in a panel of thyroid cancer cell lines. Results. We found that in most of the cell lines, although Gefitinib inhibited EGFR phosphorylation, it was poorly effective in reducing cell viability. Gefitinib, however, was able to inhibit EGF-induced cell migration and matrix invasion. In most thyroid cancer cell lines, Gefitinib significantly inhibited Akt phosphorylation by inhibiting EGFR activation, but it had limited or no effect on ERK phosphorylation. The poor cell response to Gefitinib was associated with genetic alterations leading to constitutive activation of the ERK pathway, including BRAF((V600E)) and HRAS(G12A/Q61R) mutations and RET/PTC1 rearrangement. When BRAF((V600E))-positive thyroid cancer cells were incubated with the specific BRAF inhibitor PLX4032, sensitivity to Gefitinib was restored. Similar results were obtained with RAS and RET/PTC inhibitors. Conclusions. These results indicate that thyroid cancer resistance to Gefitinib is due to the constitutive activation of the mitogenic pathway by either signals downstream of EGFR or other tyrosine kinase receptors. This resistance can be overcome by the combined use of selective inhibitors.
The Journal of Clinical Endocrinology and Metabolism 04/2013; DOI:10.1210/jc.2012-3623 · 6.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The BCR-ABL oncoprotein of Chronic Myelogenous Leukemia (CML) displays exclusive cytoplasmic localization and constitutive tyrosine kinase activity leading to the activation of different pathways that favor cell proliferation and survival. BCR-ABL induces survivin expression at both the mRNA and protein level, thus inhibiting the apoptotic machinery of CML cells and contributing to the expansion of the leukemic clone. We report that, in human CML cell lines, BCR-ABL-mediated up-regulation of survivin involves the JAK2/STAT3 pathway since silencing of either protein caused a consistent reduction in survivin expression. Cell lines unresponsive to Imatinib Mesylate (IM) because of BCR-ABL gene amplification were not resensitized to the drug after survivin down-regulation. However, cells insensitive to IM because of point mutations in the BCR-ABL kinase domain were highly responsive to Hydroxyurea (HU) after survivin silencing. To address the possible clinical applications of our results we employed shepherdin, a cell-permeable peptidomimetic compound that down-regulates survivin expression by preventing its interaction with heat shock protein 90. Incubation with shepherdin of immortalized cell lines both sensitive and resistant to IM enhanced cell death induced by HU and Doxorubicin. Similarly, the combination of shepherdin with first and second-generation tyrosine kinase inhibitors reduced the colony-forming potential of human progenitors derived from both IM-sensitive and IM-resistant CML patients. These results suggest that strategies aimed at reducing survivin levels may represent a potential therapeutic option for CML patients unresponsive to IM.
Molecular Cancer Therapeutics 03/2013; 12(6). DOI:10.1158/1535-7163.MCT-12-0550 · 6.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Treatment of Chronic Myeloid Leukemia (CML) has evolved rapidly in the last 10 years. The objectives of this national consensus meeting were to describe the optimal procedures to perform at diagnosis, the most appropriate choice of tyrosine kinase inhibitor (TKI) in the first line setting, the correct monitoring procedures, the appropriate timing for resistance identification allowing a rapid TKI switch, and the future possibility of treatment discontinuation. A panel of experts in CML management were invited for a 2-day workshop. Prior to the conference, the organizing committee selected several topics and assigned them to different physicians divided in four groups. Issues discussed were: 1) role of cytogenetic and molecular response monitoring in 2013; 2) frontline treatment of CML in 2013 and therapeutic objectives; 3) how to monitor response and when to change therapy after resistance or non-optimal responses; 4) possible therapy discontinuation after achievement of deep and stable molecular responses. Different national experts reviewed the literature, analysed levels of evidence for each topic and, after extensive discussions within smaller working groups, presented their conclusions during the meeting. Each consensus aim was then evaluated by a general vote in the plenary sessions
Critical reviews in oncology/hematology 01/2013; DOI:10.1016/j.critrevonc.2013.12.010 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To assess the impact of BCR-ABL kinase domain mutations on dasatinib response in elderly chronic myeloid leukemia (CML) patients, we analyzed the outcome of 76 individuals aged >60 affected by imatinib-resistant chronic-phase CML. We found that 36 cases (47 %) displayed mutations before dasatinib. Compared to non-mutated patients, subjects with point mutations had a worse response to dasatinib, with significantly lower rates of complete cytogenetic response (57 vs 32 %), higher percentage of primary resistance (16/36 vs 6/40) and a trend towards a shorter median event-free survival. Our data suggest that, in elderly patients, detection of BCR-ABL mutations negatively affects response to dasatinib.
Annals of Hematology 10/2012; DOI:10.1007/s00277-012-1591-2 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We read with great interest the manuscript by Baron and colleagues entitled "Value of infliximab (Remicade ®) in patients with low-risk myelodysplastic syndrome: final results of a randomized phase II trial (EORTC trial 06023) of the EORTC Leukemia Group". 1 The reported findings showed that infliximab displayed modest efficacy in patients with low-risk myelodysplastic syndrome (MDS). However, since a combination of infliximab and chemotherapy might represent a promising therapeutic strategy for MDS and acute myeloid leukemia (AML) patients, two safety issues should be considered. The first issue concerns the possible risk of secondary malignancies associated with infliximab treatment. We recently diagnosed chronic myeloid leukemia (CML) in chronic phase (high Sokal risk; e14a2, high BCR-ABL IS transcript 74.6%) in a 54-year old patient with rheuma-toid arthritis who had been taken infliximab (5 mg/kg) for five years (from 2002 to 2007). Because of his newly diag-nosed CML, he is now receiving tyrosine kinase inhibitor therapy (imatinib mesylate 400 mg daily) and has achieved a major molecular remission after 12 months. Thi is by no means an isolated case, since other accounts of lymphoproliferative or myeloproliferative disorders in patients receiving tumor necrosis alpha inhibitors have been published in the literature. 2-6 Therefore, although a direct causative link between infliximab and increased risk of second hematologic disorders has not been estab-lished, a warning should be raised. A second issue relates to the potential risk of infections in patients treated with infliximab. Baron et al. reported a high cumulative incidence of grade 3-5 infections in both arms of their study (41% of patients in the 3 mg/kg arm and 19% of patients in the 5 mg/kg arm). 1 This finding might represent a second warning signal when using infliximab in patients with hematologic malignancies because of the suppressive effect that the drug might exert on normal hematopoietic progenitors. 7 In conclusion, while infliximab might represent a promising agent with therapeutic activity in both MDS and AML, potential toxicities associated with this drug might warrant further assessment of its safety profile in specific subpopulations. Key words: myelodysplastic syndrome, acute myeloid leukemia, infliximab, secondary malignancy, infection. Citation: Stagno F, Vigneri P, Cupri A, Vitale SR, and Di Raimondo F. Infliximab therapy in hematolog malignancies: handle with care (Comment). Haematologica 2012;97(8):e26. doi:10.3324/haematol.2012.067934 The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org. Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.