Tim H. Brümmendorf

Bosutinib is active in chronic phase chronic myeloid leukemia after imatinib and dasatinib and/or nilotinib therapy failure

Bosutinib, a dual Src/Abl tyrosine kinase inhibitor (TKI), has shown potent activity against chronic myeloid leukemia (CML). This phase 1/2 study evaluated the efficacy and safety of once-daily bosutinib 500 mg in leukemia patients after resistance/intolerance to imatinib. The current analysis included 118 patients with chronic-phase CML who had been pretreated with imatinib followed by dasatinib and/or nilotinib, with a median follow-up of 28.5 months. In this subpopulation, major cytogenetic response was attained by 32% of patients; complete cytogenetic response was attained by 24%, including in one of 3 patients treated with 3 prior TKIs. Complete hematologic response was achieved/maintained in 73% of patients. On-treatment transformation to accelerated/blast phase occurred in 5 patients. At 2 years, Kaplan-Meier-estimated progression-free survival was 73% and estimated overall survival was 83%. Responses were seen across Bcr-Abl mutations, including those associated with dasatinib and nilotinib resistance, except T315I. Bosutinib had an acceptable safety profile; treatment-emergent adverse events were primarily manageable grade 1/2 gastrointestinal events and rash. Grade 3/4 nonhematologic adverse events (> 2% of patients) included diarrhea (8%) and rash (4%). Bosutinib may offer a new treatment option for patients with chronic-phase CML after treatment with multiple TKIs. This trial was registered at www.clinicaltrials.gov as NCT00261846.

Prospective study of rabbit antithymocyte globulin and cyclosporine for aplastic anemia from the EBMT Severe Aplastic Anaemia Working Party

Rabbit antithymocyte globulin (rATG; thymoglobulin, Genzyme) in combination with cyclosporine, as first-line immunosuppressive therapy, was evaluated prospectively in a multicenter, European, phase 2 pilot study, in 35 patients with aplastic anemia. Results were compared with 105 age- and disease severity-matched patients from the European Blood and Marrow Transplant registry, treated with horse ATG (hATG; lymphoglobulin) and cyclosporine. The primary end point was response at 6 months. At 3 months, no patients had achieved a complete response to rATG. Partial response occurred in 11 (34%). At 6 months, complete response rate was 3% and partial response rate 37%. There were 10 deaths after rATG (28.5%) and 1 after subsequent HSCT. Infections were the main cause of death in 9 of 10 patients. The best response rate was 60% for rATG and 67% for hATG. For rATG, overall survival at 2 years was 68%, compared with 86% for hATG (P = .009). Transplant-free survival was 52% for rATG and 76% for hATG (P = .002). On multivariate analysis, rATG (hazard ratio = 3.9, P = .003) and age more than 37 years (hazard ratio = 4.7, P = .0008) were independent adverse risk factors for survival. This study was registered at www.clinicaltrials.gov as NCT00471848.

Bosutinib safety and management of toxicity in leukemia patients with resistance or intolerance to imatinib and other tyrosine kinase inhibitors

Bosutinib is an oral, dual SRC/ABL tyrosine kinase inhibitor (TKI) with clinical activity in Philadelphia chromosome-positive (Ph(+)) leukemia. We assessed the safety and tolerability of bosutinib 500 mg per day in a phase 1/2 study in chronic-phase (CP) chronic myeloid leukemia (CML) or advanced Ph(+) leukemia following resistance/intolerance to imatinib and possibly other TKIs. Patient cohorts included second-line CP CML (n = 286), third-/fourth-line CP CML (n = 118), and advanced leukemia (n = 166). Median bosutinib duration was 11.1 (range, 0.03-83.4) months. Treatment-emergent adverse events (TEAEs) in each cohort were primarily gastrointestinal (diarrhea [86%/83%/74%], nausea [46%/48%/48%], and vomiting [37%/38%/43%]). Diarrhea presented early, with few (8%) patients experiencing grade 3/4 events; dose reduction due to diarrhea occurred in 6% of affected patients. Grade 3/4 myelosuppression TEAEs were reported in 41% of patients; among affected patients, 46% were managed with bosutinib interruption and 32% with dose reduction. Alanine aminotransferase elevation TEAEs occurred in 17% of patients (grade 3/4, 7%); among patients managed with dose interruption, bosutinib rechallenge was successful in 74%. Bosutinib demonstrated acceptable safety with manageable toxicities in Ph(+) leukemia. This trial (NCT00261846) was registered at www.ClinicalTrials.gov (this manuscript is based on a different data snapshot from that in ClinicalTrials.gov).

JAK Inhibition Impairs NK Cell Function in Myeloproliferative Neoplasms

Ruxolitinib is a small-molecule inhibitor of the JAK kinases, which has been approved for the treatment of myelofibrosis, a rare myeloproliferative neoplasm (MPN), but clinical trials are also being conducted in inflammatory-driven solid tumors. Increased infection rates have been reported in ruxolitinib-treated patients, and natural killer (NK) cells are immune effector cells known to eliminate both virus-infected and malignant cells. On this basis, we sought to compare the effects of JAK inhibition on human NK cells in a cohort of 28 MPN patients with or without ruxolitinib treatment and 24 healthy individuals. NK cell analyses included cell frequency, receptor expression, proliferation, immune synapse formation, and cytokine signaling. We found a reduction in NK cell numbers in ruxolitinib-treated patients that was linked to the appearance of clinically relevant infections. This reduction was likely due to impaired maturation of NK cells, as reflected by an increased ratio in immature to mature NK cells. Notably, the endogenous functional defect of NK cells in MPN was further aggravated by ruxolitinib treatment. In vitro data paralleled these in vivo results, showing a reduction in cytokine-induced NK cell activation. Further, reduced killing activity was associated with an impaired capacity to form lytic synapses with NK target cells. Taken together, our findings offer compelling evidence that ruxolitinib impairs NK cell function in MPN patients, offering an explanation for increased infection rates and possible long-term side effects associated with ruxolitinib treatment.

Pluripotent stem cells escape from senescence-associated DNA methylation changes

Pluripotent stem cells evade replicative senescence, whereas other primary cells lose their proliferation and differentiation potential after a limited number of cell divisions, and this is accompanied by specific senescence-associated DNA methylation (SA-DNAm) changes. Here, we investigate SA-DNAm changes in mesenchymal stromal cells (MSC) upon long-term culture, irradiation-induced senescence, immortalization, and reprogramming into induced pluripotent stem cells (iPSC) using high-density HumanMethylation450 BeadChips. SA-DNAm changes are highly reproducible and they are enriched in intergenic and nonpromoter regions of developmental genes. Furthermore, SA-hypomethylation in particular appears to be associated with H3K9me3, H3K27me3, and Polycomb-group 2 target genes. We demonstrate that ionizing irradiation, although associated with a senescence phenotype, does not affect SA-DNAm. Furthermore, overexpression of the catalytic subunit of the human telomerase (TERT) or conditional immortalization with a doxycycline-inducible system (TERT and SV40-TAg) result in telomere extension, but do not prevent SA-DNAm. In contrast, we demonstrate that reprogramming into iPSC prevents almost the entire set of SA-DNAm changes. Our results indicate that long-term culture is associated with an epigenetically controlled process that stalls cells in a particular functional state, whereas irradiation-induced senescence and immortalization are not causally related to this process. Absence of SA-DNAm in pluripotent cells may play a central role for their escape from cellular senescence.

Safety of bosutinib versus imatinib in the phase 3 BELA trial in newly diagnosed chronic phase chronic myeloid leukemia

Bosutinib, an orally active, Src/Abl tyrosine kinase inhibitor, has demonstrated clinical activity and acceptable tolerability in chronic phase chronic myeloid leukemia (CP CML). This updated analysis of the BELA trial assessed the safety profile and management of toxicities of bosutinib versus imatinib in adults with newly diagnosed (≤6 months) CP CML after >30 months from accrual completion. Among patients randomized to bosutinib 500 mg/d (n = 250) or imatinib 400 mg/d (n = 252), 248 and 251, respectively, received ≥1 dose of study treatment. Adverse events (AEs; any grade) with bosutinib versus imatinib were significantly more common for certain gastrointestinal events (diarrhea, 70% vs. 26%; P < 0.001; vomiting, 33% vs. 16%; P < 0.001), alanine aminotransferase (33% vs. 9%; P < 0.001) and aspartate aminotransferase (28% vs. 10%; P < 0.001) elevations, and pyrexia (19% vs. 12%; P = 0.046). AEs significantly less common with bosutinib included edema (periorbital, 2% vs. 14%; P < 0.001; peripheral, 5% vs. 12%; P = 0.006), musculoskeletal (myalgia, 5% vs. 12%; P = 0.010; muscle cramps, 5% vs. 22%; P < 0.001; bone pain, 4% vs. 11%; P = 0.003), increased creatine phosphokinase (8% vs. 20%; P < 0.001), neutropenia (13% vs. 30%; P < 0.001), and leukopenia (9% vs. 22%; P < 0.001). Between‐group differences in the incidence of cardiac and vascular AEs were not significant. Diarrhea was typically transient, mostly Grade 1/2, occurring early during treatment, and was manageable with antidiarrheal medication. Despite higher rates of aminotransferase elevation with bosutinib, events were managed in most patients with dose modification and/or concomitant medication. Bosutinib had a manageable safety profile distinct from that of imatinib in patients with newly diagnosed CP CML. Am. J. Hematol. 89:947–953, 2014.

Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.

Rps14 haploinsufficiency causes a block in erythroid differentiation mediated by S100A8 and S100A9.

Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8—whose expression was increased in mutant erythroblasts, monocytes and macrophages—is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation

BackgroundWe have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways.ResultsMethylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2.ConclusionFrom these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.

Current Status and Perspectives of Tyrosine Kinase Inhibitor Treatment in the Posttransplant Period in Patients with Chronic Myelogenous Leukemia (CML)

Following the introduction of tyrosine kinase inhibitors (TKIs) in chronic myelogenous leukemia (CML), allogeneic stem cell transplantation (SCT) took a shift toward high-risk patients. Considering the high relapse rates posttransplant in these selected patients, several studies evaluated posttransplant use of the TKI imatinib. Although the number of studies are still limited, and data have to be confirmed by additional studies, safety of imatinib even within the first months after SCT seems to be acceptable. Imatinib was shown to be effective in patients with molecular or hematologic relapse of chronic or accelerated phase posttransplant (CP, AP), whereas outcomes in blast phase were more unfavorable. The compound further seemed beneficial for prophylactic use in patients who achieved complete remission posttransplant. The combination of imatinib with donor lymphocytes did not result in increased toxicity or graft-versus-host disease (GVHD). First studies suggest that second-generation TKIs such as dasatinib or nilotinib are manageable posttransplant with acceptable toxicity as well. In conclusion, TKIs of the first- and second-generation are promising options for the posttransplant period of patients with CML, but algorithms for dosage, intervals from SCT, duration of application, and the combination with donor lymphocytes still have to be developed.