Sebastian Scholl

High activity of sorafenib in FLT3-ITD-positive acute myeloid leukemia synergizes with allo-immune effects to induce sustained responses

Preliminary evidence suggests that the multikinase inhibitor sorafenib has clinical activity in FLT3-ITD-positive (FLT3-ITD) acute myeloid leukemia (AML). However, the quality and sustainability of achievable remissions and clinical variables that influence the outcome of sorafenib monotherapy are largely undefined. To address these questions, we evaluated sorafenib monotherapy in 65 FLT3-ITD AML patients treated at 23 centers. All but two patients had relapsed or were chemotherapy-refractory after a median of three prior chemotherapy cycles. Twenty-nine patients (45%) had undergone prior allogeneic stem cell transplantation (allo-SCT). The documented best responses were: hematological remission in 24 patients (37%), bone marrow remission in 5 patients (8%), complete remission (with and without normalization of peripheral blood counts) in 15 patients (23%) and molecular remission with undetectable FLT3-ITD mRNA in 10 patients (15%), respectively. Seventeen of the patients without prior allo-SCT (47%) developed sorafenib resistance after a median treatment duration of 136 days (range, 56–270 days). In contrast, allo-SCT patients developed sorafenib resistance less frequently (38%) and significantly later (197 days, range 38–225 days; P=0.03). Sustained remissions were seen exclusively in the allo-SCT cohort. Thus, sorafenib monotherapy has significant activity in FLT3-ITD AML and may synergize with allogeneic immune effects to induce durable remissions.

Clinical impact of nucleophosmin mutations and Flt3 internal tandem duplications in patients older than 60 yr with acute myeloid leukaemia

Background:  Nucleophosmin (NPM1) and Flt3 internal tandem duplications (Flt3‐ITD mutations) represent the most frequent molecular aberrations in patients with acute myeloid leukemia (AML). While NPM1 mutations are associated with favourable prognosis in younger AML patients, Flt3‐ITD mutations reflect an unfavourable prognostic factor in these patients. So far, especially NPM1 mutations have not yet been evaluated exclusively in older patients.

Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ

Signal transduction of FMS-like tyrosine kinase 3 (FLT3) is regulated by protein-tyrosine phosphatases (PTPs). We recently identified the PTP DEP-1/CD148/PTPRJ as a novel negative regulator of FLT3. This study addressed the role of DEP-1 for regulation of the acute myeloid leukemia (AML)-related mutant FLT3 internal tandem duplication (ITD) protein. Our experiments revealed that DEP-1 was expressed but dysfunctional in cells transformed by FLT3 ITD. This was caused by enzymatic inactivation of DEP-1 through oxidation of the DEP-1 catalytic cysteine. In intact cells, including primary AML cells, FLT3 ITD kinase inhibition reactivated DEP-1. DEP-1 reactivation was also achieved by counteracting the high levels of reactive oxygen species (ROS) production detected in FLT3 ITD-expressing cell lines by inhibition of reduced NAD phosphate (NADPH)-oxidases, or by overexpression of catalase or peroxiredoxin-1 (Prx-1). Interference with ROS production in 32D cells inhibited cell transformation by FLT3 ITD in a DEP-1-dependent manner, because RNAi-mediated depletion of DEP-1 partially abrogated the inhibitory effect of ROS quenching. Reactivation of DEP-1 by stable overexpression of Prx-1 extended survival of mice in the 32D cell/C3H/HeJ mouse model of FLT3 ITD-driven myeloproliferative disease. The study thus uncovered DEP-1 oxidation as a novel event contributing to cell transformation by FLT3 ITD.

Ponatinib may overcome resistance of FLT3-ITD harbouring additional point mutations, notably the previously refractory F691I mutation.

Fms‐like tyrosine kinase (FLT3) mutations are the most frequent mutations in patients with acute myeloid leukaemia (AML) that confer a poor prognosis. Constitutively active FLT3‐ITD (internal tandem duplications) mutations define a promising target for therapeutic approaches using small molecule inhibitors. However, several point mutations of the FLT3 tyrosine kinase domain (FLT3‐TKD) have been identified to mediate resistance towards FLT3 tyrosine kinase inhibitors (FLT3‐TKI), including secondary mutations of FLT3. We investigated the cellular effects of the recently characterised FLT3‐TKI ponatinib (AP24534) on murine myeloid cells transfected with FLT3‐ITD with or without additional point mutations of the FLT3‐TKD including the (so far) multi‐resistant F691I mutation. Ponatinib effectively induced apoptosis not only in the parental FLT3‐ITD cell line but also in all stably transfected subclones harbouring additional FLT3‐TKD point mutations (N676D, F691I or G697R). These observations correlated with a strong inhibition of FLT3‐ITD and its downstream targets STAT5, AKT and ERK1/2 upon ponatinib incubation, as determined by Western blotting. We conclude that ponatinib represents a promising FLT3‐TKI that should be further investigated in clinical trials. The targeted therapy of FLT3‐ITD‐positive AML with ponatinib might be associated with a lower frequency of secondary resistance caused by acquired FLT3‐TKD mutations.

Clinical implications of molecular genetic aberrations in acute myeloid leukemia

The role of different cytogenetic changes has been extensively evaluated in patients with acute myeloid leukemia (AML), and cytogenetic analysis of AML blasts is essential to form prognostic subgroups in order to stratify for the extent of therapy. Nevertheless, 40–45% of AML patients lack such cytogenetic markers, i.e., cytogenetically normal AML (CN-AML). In the past decade, different molecular aberrations were identified in AML and especially CN-AML can now be discriminated into certain prognostic subgroups. This review considers the latest advances to define the prognostic impact of molecular aberrations in AML and gives insights how such molecular markers can be applied for analysis of minimal residual disease. Furthermore, therapeutic implications as well as the potential role of new methodological techniques in analyzing expression patterns of AML blasts are discussed.

NOX4-driven ROS formation mediates PTP inactivation and cell transformation in FLT3ITD-positive AML cells

Activating mutations of FMS-like tyrosine kinase 3 (FLT3), notably internal tandem duplications (ITDs), are associated with a grave prognosis in acute myeloid leukemia (AML). Transforming FLT3ITD signal transduction causes formation of reactive oxygen species (ROS) and inactivation of the protein-tyrosine phosphatase (PTP) DEP-1/PTPRJ, a negative regulator of FLT3 signaling. Here we addressed the underlying mechanisms and biological consequences. NADPH oxidase 4 (NOX4) messenger RNA and protein expression was found to be elevated in FLT3ITD-positive cells and to depend on FLT3ITD signaling and STAT5-mediated activation of the NOX4 promoter. NOX4 knockdown reduced ROS levels, restored DEP-1 PTP activity and attenuated FLT3ITD-driven transformation. Moreover, Nox4 knockout (Nox4−/−) murine hematopoietic progenitor cells were refractory to FLT3ITD-mediated transformation in vitro. Development of a myeloproliferative-like disease (MPD) caused by FLT3ITD-transformed 32D cells in C3H/HeJ mice, and of a leukemia-like disease in mice transplanted with MLL-AF9/ FLT3ITD-transformed murine hematopoietic stem cells were strongly attenuated by NOX4 downregulation. NOX4-targeting compounds were found to counteract proliferation of FLT3ITD-positive AML blasts and MPD development in mice. These findings reveal a previously unrecognized mechanism of oncoprotein-driven PTP oxidation, and suggest that interference with FLT3ITD-STAT5-NOX4-mediated overproduction of ROS and PTP inactivation may have therapeutic potential in a subset of AML.

Polymorphisms of Toll-like receptors (TLR2 and TLR4) are associated with the risk of infectious complications in acute myeloid leukemia.

Infectious complications continue to be one of the major causes of morbidity and mortality in patients with acute myeloid leukemia (AML). Several single-nucleotide polymorphisms (SNPs) of Toll-like receptors (TLRs) can affect the genetic susceptibility to infections or even sepsis. We sought to investigate the impact of different SNPs on the incidence of developing sepsis and pneumonia in patients with newly diagnosed AML following induction chemotherapy. We analyzed three SNPs in the TLR2 (Arg753Gln) and TLR4 (Asp299Gly and Thr399Ile) gene in a cohort of 155 patients with AML who received induction chemotherapy. The risk of developing sepsis and pneumonia was assessed by multiple logistic regression analyses. The presence of the TLR2 Arg753Gln polymorphism was significantly associated with pneumonia in AML patients (odds ratio (OR): 10.78; 95% confidence interval (CI): 2.0–58.23; P=0.006). Furthermore, the cosegregating TLR4 polymorphisms Asp299Gly and Thr399Ile were independent risk factors for the development of both sepsis and pneumonia (OR: 3.55; 95% CI: 1.21–10.4, P=0.021 and OR: 3.57, 95% CI: 1.3–9.86, P=0.014, respectively). To our best knowledge, this study represents the first analysis demonstrating that polymorphisms of TLR2 and TLR4 influence the risk of infectious complications in patients with AML undergoing induction chemotherapy.

Safety and impact of donor‐type red blood cell transfusion before allogeneic peripheral blood progenitor cell transplantation with major ABO mismatch

BACKGROUND:  Changes within the ABO system are regularly observed phenomena in allogeneic bone marrow transplantation (BMT) and peripheral blood progenitor cell transplantation (PBPCT). Major ABO mismatch can lead to different clinical problems including acute hemolysis after infusion of the allograft, delay of red blood cell (RBC) engraftment, or even manifestation of pure red cell aplasia (PRCA).

Paraneoplastic inflammation in myelodysplastic syndrome or bone marrow failure: case series with focus on 5‐azacytidine and literature review

Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal disorders of haematopoietic stem cells, characterised by dysplastic haematopoiesis and dysregulated apoptosis resulting in various degrees of cytopenia, whereas canonical cytologic, cytogenetic and histopathologic findings guiding the diagnosis MDS are widely accepted, the MDS‐phenotype can be masked by coexisting/paraneoplastic immunologic disease. Autoimmune disorders have an estimated incidence of 10% among patients suffering from MDS and are causally related to increased morbidity and mortality, younger age at diagnosis and more complex genetics. Conversely, systemic inflammatory disorders may be an early manifestation of MDS, show good response to immunosuppressive therapy and frequently disappear during the course of specific haematologic therapy.

Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells: FLT3 ITD sustains high levels of DUSP6 expression

Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation.