Ruxolitinib Adherence in Myelofibrosis and Polycythemia Vera: the "RAMP" Italian multicenter prospective study.

Ruxolitinib is beneficial in patients with myelofibrosis (MF) and polycythemia vera (PV). Information on ruxolitinib adherence is scant. The Ruxolitinib Adherence in Myelofibrosis and Polycythemia Vera (RAMP) prospective multicenter study (NCT06078319) included 189 ruxolitinib-treated patients. Patients completed the Adherence to Refills and Medications Scale (ARMS) and Distress Thermometer and Problem List (DTPL) at the earliest convenience, after registration in the study, and at later timepoints. At week-0, low adherence (ARMS > 14) and high distress (DT ≥ 4) were declared by 49.7% and 40.2% of patients, respectively. The main reason for low adherence was difficult ruxolitinib supply (49%), intentional (4.3%) and unintentional (46.7%) non-take. In multivariable regression analysis, low adherence was associated to male sex (p = 0.001), high distress (p < 0.001), and treatment duration ≥ 1 year (p = 0.03). Over time, rates of low adherence and high distress remained stable, but unintentional non-take decreased from 47.9% to 26.0% at week-48. MF patients with stable high adherence/low distress were more likely to obtain/maintain the spleen response at week-24. Low adherence to ruxolitinib represents an unmet clinical need that require a multifaceted approach, based on reason behind it (patients characteristics and treatment duration). Its recognition may help distinguishing patients who are truly refractory and those in need of therapy optimization.

Pomalidomide in myeloproliferative neoplasm-associated myelofibrosis.

Myeloproliferative neoplasm (MPN)-associated myelofibrosis is a MPN characterized by bone marrow fibrosis, cytopenias, splenomegaly and constitutional symptoms. Pomalidomide, an immune-modifying drug, is reported to improve anaemia and thrombocytopenia in some patients with MPN-associated myelofibrosis. We designed a phase 2 study of pomalidomide in patients with MPN-associated myelofibrosis and anaemia and/or thrombocytopenia and/or neutropenia. Subjects received pomalidomide 2.0 mg/day in cohort 1 (n=38) or 0.5 mg/day in cohort 2 (n=58). Prednisolone was added if there was no response after 3 months in cohort 1 and based on up-front randomization in cohort 2 if there was no response at 3 or 6 months. Response rates were 39% (95% confidence interval (CI), 26-55%) in cohort 1 and 24% (95% CI, 15-37%) in cohort 2. In a multivariable logistic regression model pomalidomide at 2.0 mg/day (odds ratio (OR), 2.62; 95% CI, 1.00-6.87; P=0.05) and mutated TET2 (OR, 5.07; 95% CI, 1.16-22.17; P=0.03) were significantly associated with responses. Median duration of responses was 13.0 months (range 0.9-52.7). There was no significant difference in response rates or duration in subjects receiving or not receiving prednisolone. Clinical trial MPNSG 01-09 is registered at ClinicalTrials.gov (NCT00949364) and clinicaltrialsregister.eu (EudraCT Number: 2009-010738-23).

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.

Epo-induced erythroid maturation is dependent on Plcγ1 signaling.

Erythropoiesis is a tightly regulated process. Development of red blood cells occurs through differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Binding of erythropoietin (Epo) to its receptor (EpoR) is required for erythropoiesis as it promotes survival and late maturation of erythroid progenitors. In vivo and in vitro studies have highlighted the requirement of EpoR signaling through Janus kinase 2 (Jak2) tyrosine kinase and Stat5a/b as a central pathway. Here, we demonstrate that phospholipase C gamma 1 (Plcγ1) is activated downstream of EpoR-Jak2 independently of Stat5. Plcγ1-deficient pro-erythroblasts and erythroid progenitors exhibited strong impairment in differentiation and colony-forming potential. In vivo, suppression of Plcγ1 in immunophenotypically defined HSCs (Lin(-)Sca1(+)KIT(+)CD48(-)CD150(+)) severely reduced erythroid development. To identify Plcγ1 effector molecules involved in regulation of erythroid differentiation, we assessed changes occurring at the global transcriptional and DNA methylation level after inactivation of Plcγ1. The top common downstream effector was H2afy2, which encodes for the histone variant macroH2A2 (mH2A2). Inactivation of mH2A2 expression recapitulated the effects of Plcγ1 depletion on erythroid maturation. Taken together, our findings identify Plcγ1 and its downstream target mH2A2, as a 'non-canonical' Epo signaling pathway essential for erythroid differentiation.

Targeting MCL-1 sensitizes FLT3-ITD-positive leukemias to cytotoxic therapies.

Patients suffering from acute myeloid leukemias (AML) bearing FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) have poor outcomes following cytarabine- and anthracyclin-based induction therapy. To a major part this is attributed to drug resistance of FLT3-ITD-positive leukemic cells. Against this background, we have devised an antibody array approach to identify proteins, which are differentially expressed by hematopoietic cells in relation to activated FLT3 signaling. Selective upregulation of antiapoptotic myeloid cell leukemia-1 (MCL-1) was found in FLT3-ITD-positive cell lines and primary mononuclear cells from AML patients as compared with FLT3-wild-type controls. Upregulation of MCL-1 was dependent on FLT3 signaling as confirmed by its reversion upon pharmacological inhibition of FLT3 activity by the kinase inhibitor PKC412 as well as siRNA-mediated suppression of FLT3. Heterologously expressed MCL-1 substituted for FLT3 signaling by conferring resistance of hematopoietic cells to antileukemia drugs such as cytarabine and daunorubicin, and to the proapoptotic BH3 mimetic ABT-737. Conversely, suppression of endogenous MCL-1 by siRNA or by flavopiridol treatment sensitized FLT3-ITD-expressing hematopoietic cells to cytotoxic and targeted therapeutics. In conclusion, MCL-1 is an essential effector of FLT3-ITD-mediated drug resistance. Therapeutic targeting of MCL-1 is a promising strategy to overcome drug resistance in FLT3-ITD-positive AML.

Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway.

In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.

Biased distribution of chromosomal breakpoints involving the MLL gene in infants versus children and adults with t(4;11) ALL.

Derivative chromosomes of 40 patients diagnosed with t(4;11) acute lymphoblastic leukemia (ALL) were analysed on the genomic DNA level. Chromosomal breakpoints were identified in most cases within the known breakpoint cluster regions of the involved MLL and AF4 genes. Due to our current knowledge of the primary DNA sequences of both breakpoint cluster regions, specific features were identified at the chromosomal fusion sites, including deletions, inversions and duplications of parental DNA sequences. After separation of all t(4;11) leukemia patients into two age classes (below and above 1 year of age), the analysis of chromosomal fusion sites revealed significant differences in the distribution of chromosomal breakpoints and led to the definition of two hotspot areas within the MLL breakpoint cluster region. This may point to the possibility of different age-linked mechanisms that were leading to t(4;11) chromosomal translocations.