MDM2- and FLT3-inhibitors in the treatment of FLT3-ITD acute myeloid leukemia, specificity and efficacy of NVP-HDM201 and midostaurin.

Prognosis for FLT3-ITD positive acute myeloid leukemia with high allelic ratio (>0.5) is poor, particularly in relapse, refractory to or unfit for intensive treatment, thus highlighting an unmet need for novel therapeutic approaches. The combined use of compounds targeting both the mutated FLT3 receptor and cellular p53 inhibitors might be a promising treatment option for this poor risk leukemia subset. We therefore assessed MDM2 and FLT3 inhibitors as well as cytotoxic compounds used for conventional induction treatment as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. Acute myeloid leukemia cells represented all major morphologic and molecular subtypes with normal karyotype, including FLT3-ITD (>0.5) and FLT3 wild type, NPM1 mutant and NPM1 wild type, as well as TP53 mutant and TP53 wild type cell lines. Acute myeloid leukemia cells with mutated or deleted TP53 were resistant to MDM2- and FLT3-inhibitors. FLT3-ITD positive TP53 wild type acute myeloid leukemia cells were significantly more susceptible to FLT3-inhibitors than FLT3-ITD negative TP53 wild type cells. The presence of a NPM1 mutation reduced the susceptibility of TP53 wild type acute myeloid leukemia cells to the MDM2 inhibitor NVP-HDM201. Moreover, the combined use of MDM2- and FLT3-inhibitors was superior to single agent treatment, and the combination of midostaurin and NVP-HDM201 was as specific and effective against FLT3-ITD positive TP53 wild type cells as the combination of midostaurin with conventional induction therapy. In summary, the combined use of the MDM2 inhibitor NVP-HDM201 and the FLT3 inhibitor midostaurin was a most effective and specific treatment to target TP53 and NPM1 wild type acute myeloid leukemia cells with high allelic FLT3-ITD ratio. These data suggest that the combined use of NVP-HDM201 and midostaurin might be a promising treatment option particularly in FLT3-ITD positive acute myeloid leukemia relapsed or refractory to conventional therapy.

Adverse outcome of AML with aberrant CD16 and CD56 NK cell marker expression.

Natural killer cells mediate antibody-dependent cell-mediated cytotoxicity, and CD16 exerts key functions to induce antibody-dependent cell-mediated cytotoxicity response. Because the prognostic relevance of aberrant CD16 expression in AML patients at diagnosis is unknown, we analyzed 325 AML patients undergoing intensive chemotherapy for aberrant CD16+ and CD56+ natural killer-cell marker expression. CD56+ AML patients had inferior median event-free (EFS; P = 0.0699) and overall survival (OS; 10.9 versus 20.6 months; P = 0.0132). Patients expressing CD16 had worse median EFS (P = 0.0622) and OS (13.0 versus 45.9 months; P = 0.0277). EFS for CD16+/CD56+ patients was 5.7 months compared with 7.1 months for CD16-/CD56- (P = 0.3690), and OS was 10.6 months for CD16+/CD56+ patients compared with 52.2 months for CD16-/CD56- patients (P = 0.0311). Patients with CD16+/CD56+ expression had a lower probability to achieve complete remission after 2 induction cycles (52% versus 72%). Our data suggest that AML patients with aberrant CD16 and CD56 expression have adverse survival outcomes.

Dose-intensified bendamustine and melphalan (BenMel) conditioning before second autologous transplantation in myeloma patients.

Consolidation in myeloma patients with high-dose melphalan chemotherapy (Mel HDCT) and autologous transplantation (ASCT) is standard of care since more than 2 decades. However, definite cure remains exceptional despite intensive treatment, and improving effectiveness of HDCT remains an unmet clinical need. Combining intensified bendamustine with melphalan may represent an option. We analyzed safety and efficacy of combining dose-intensified bendamustine (200 mg/m2 on days -4/-3) with high-dose melphalan (100 mg/m2 on days -2/-1) before a second (tandem) ASCT in adverse risk myeloma patients after Mel HDCT/ASCT1. Twelve patients received BenMel conditioning before ASCT2 because of high-risk cytogenetics and/or failure to achieve complete remission (CR) after Mel HDCT/ASCT1. Comparing Mel HDCT/ASCT1 and BenMel HDCT/ASCT2, we observed no differences in hematologic recovery and tolerance. Acute renal injury after BenMel conditioning occurred in 3 (25%) patients, but was reversible in all patients, and there were no treatment related deaths. Complete remission rates were increasing from 42% after Mel/ASCT1 to 75% after BenMel/ASCT2. PFS 1 year after ASCT2 was 67%, and OS was 83%. These data suggest that dose-intensified bendamustine with melphalan conditioning is safe and warrants a prospective randomized comparison to standard melphalan HDCT in myeloma patients.

Iron overload is correlated with impaired autologous stem cell mobilization and survival in acute myeloid leukemia.

BACKGROUND: Patients with acute myeloid leukemia (AML) undergoing consolidation with autologous stem cell transplantation (ASCT) depend on the successful mobilization of peripheral blood stem cells. However, the factors affecting the mobilization potential in AML patients and, in particular, the effect of transfusion-related iron overload on peripheral blood stem cell mobilization are largely unknown. STUDY DESIGN AND METHODS: We investigated the association of varying levels of iron overload and stem cell mobilization efficacy in consecutive AML patients after two induction cycles. RESULTS: A total of 113 AML patients in early first complete remission underwent the mobilization procedure. While 84 (74.3%) patients had serum ferritin levels exceeding 1000 µg/L, 26 (23.0%) patients had levels even higher than 2000 µg/L. Iron overload correlated with the number of preceding red blood cell transfusions and inversely correlated with circulating CD34+ cell levels (p = 0.04) at apheresis. Finally, the median progression-free and overall survival rates of patients with ferritin levels of higher than 2000 µg/L were shorter with 332 days versus 2156 days (p = 0.04) and 852 days versus 2235 days (p = 0.04), respectively. CONCLUSION: Our data suggest that transfusion-related iron overload is suppressing the mobilization potential and is associated with inferior outcome in AML.

Zevalin and BEAM (Z-BEAM) versus rituximab and BEAM (R-BEAM) conditioning chemotherapy prior to autologous stem cell transplantation in patients with mantle cell lymphoma.

Early relapse is common in patients with mantle cell lymphoma (MCL) highlighting the unmet need for further improvement of therapeutic options for these patients. CD20 inhibition combined with induction chemotherapy as well as consolidation with high-dose chemotherapy (HDCT) is increasingly considered cornerstones within current therapy algorithms of MCL whereas the role of radioimmunotherapy is unclear. This retrospective single center study compared 46 consecutive MCL patients receiving HDCT in first or second remission. Thirty-five patients had rituximab and BEAM (R-BEAM), and 11 patients received ibritumomab tiuxetan (Zevalin®), an Yttrium-90 labeled CD20 targeting antibody, prior to BEAM (Z-BEAM) followed by autologous stem cell transplantation (ASCT). We observed that the 5-year overall survival (OS) in the R-BEAM and Z-BEAM groups was 55% and 71% (p = 0.288), and the 4-year progression free survival (PFS) was 32% and 41%, respectively (p = 0.300). There were no treatment related deaths in both groups, and we observed no differences in toxicities, infection rates or engraftment. Our data suggest that the Z-BEAM conditioning regimen followed by ASCT is well tolerated, but was not associated with significantly improved survival compared to R-BEAM. Copyright © 2015 John Wiley & Sons, Ltd.

Delayed Haematological recovery after autologous stem cell transplantation is associated with favourable outcome in acute myeloid leukaemia.

Autologous stem cell transplantation (ASCT) is applied to consolidate first remission in patients with acute myeloid leukaemia (AML). However, outcome after ASCT widely varies among AML patients. We analyzed the prognostic significance of haematological recovery for neutrophils [absolute neutrophil count (ANC) >1·0 × 10(9) /l] and platelets (platelet count >20·0 × 10(9) /l), stratifying at day 20 after ASCT in 88 consecutive and homogeneously treated AML patients in first remission. We observed that patients with delayed recovery had better overall survival (OS; ANC: P < 0·0001 and platelets: P = 0·0062) and time to progression (TTP; ANC: P = 0·0003 and platelets: P = 0·0125). Delayed recovery was an independent marker for better OS and TTP in a multivariate analysis including age, gender, number of transfused CD34+ cells, cytogenetics, FLT3-internal tandem duplication and NPM1 mutation. Our results suggest that delayed neutrophil and platelet recovery is associated with longer OS and TTP in AML patients consolidated with ASCT in first remission.

The angiogenesis inhibitor vasostatin is regulated by neutrophil elastase-dependent cleavage of calreticulin in AML patients.

The calcium-binding protein calreticulin (CRT) regulates protein folding in the endoplasmic reticulum (ER) and is induced in acute myeloid leukemia (AML) cells with activation of the unfolded protein response. Intracellular CRT translocation to the cell surface induces immunogenic cell death, suggesting a role in tumor suppression. In this study, we investigated CRT regulation in the serum of patients with AML. We found that CRT is not only exposed by exocytosis on the outer cell membrane after treatment with anthracyclin but also ultimately released to the serum in vitro and in AML patients during induction therapy. Leukemic cells of 113 AML patients showed increased levels of cell-surface CRT (P < .0001) and N-terminus serum CRT (P < .0001) compared with normal myeloid cells. Neutrophil elastase was identified to cleave an N-terminus CRT peptide, which was characterized as vasostatin and blocked ATRA-triggered differentiation. Levels of serum vasostatin in patients with AML inversely correlated with bone marrow vascularization, suggesting a role in antiangiogenesis. Finally, patients with increased vasostatin levels had longer relapse-free survival (P = .04) and specifically benefited from autologous transplantation (P = .006). Our data indicate that vasostatin is released from cell-surface CRT and impairs differentiation of myeloid cells and vascularization of the bone marrow microenvironment.

Deficient CEBPA DNA binding function in normal karyotype AML patients is associated with favorable prognosis.

CCAAT/enhancer binding protein-α (CEBPA) mutations in acute myeloid leukemia (AML) patients with a normal karyotype (NK) confer favorable prognosis, whereas NK-AML patients per se are of intermediate risk. This suggests that blocked CEBPA function characterizes NK-AML with favorable outcome. We determined the prognostic significance of CEBPA DNA binding function by enzyme-linked immunosorbent assay in 105 NK-AML patients. Suppressed CEBPA DNA binding was defined by 21 good-risk AML patients with inv(16) or t(8;21) (both abnormalities targeting CEBPA) and 8 NK-AML patients with dominant-negative CEBPA mutations. NK-AML patients with suppressed CEBPA function showed a better overall survival (P = .0231) and disease-free survival (P = .0069) than patients with conserved CEBPA function. Suppressed CEBPA DNA binding was an independent marker for better overall survival and disease-free survival in a multivariable analysis that included FLT3-ITD, NPM1 and CEBPA mutation status, white blood cell count, age and lactate dehydrogenase. These data indicate that suppressed CEBPA function is associated with favorable prognosis in NK-AML patients.

Protein disulfide isomerase blocks CEBPA translation and is up-regulated during the unfolded protein response in AML.

Deregulation of the myeloid key transcription factor CEBPA is a common event in acute myeloid leukemia (AML). We previously reported that the chaperone calreticulin is activated in subgroups of AML patients and that calreticulin binds to the stem loop region of the CEBPA mRNA, thereby blocking CEBPA translation. In this study, we screened for additional CEBPA mRNA binding proteins and we identified protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) resident protein, to bind to the CEBPA mRNA stem loop region. We found that forced PDI expression in myeloid leukemic cells in fact blocked CEBPA translation, but not transcription, whereas abolishing PDI function restored CEBPA protein. In addition, PDI protein displayed direct physical interaction with calreticulin. Induction of ER stress in leukemic HL60 and U937 cells activated PDI expression, thereby decreasing CEBPA protein levels. Finally, leukemic cells from 25.4% of all AML patients displayed activation of the unfolded protein response as a marker for ER stress, and these patients also expressed significantly higher PDI levels. Our results indicate a novel role of PDI as a member of the ER stress-associated complex mediating blocked CEBPA translation and thereby suppressing myeloid differentiation in AML patients with activated unfolded protein response (UPR).

Heterozygous deletion of the PU.1 locus in human AML.

The transcription factor PU.1 is essential for myeloid development. Targeted disruption of an upstream regulatory element (URE) decreases PU.1 expression by 80% and leads to acute myeloid leukemia (AML) in mice. Here, we sequenced the URE sequences of PU.1 in 120 AML patients. Four polymorphisms (single nucleotide polymorphisms [SNPs]) in the URE were observed, with homozygosity in all SNPs in 37 patients. Among them, we compared samples at diagnosis and remission, and one patient with cytogenetically normal acute myeloid leukemia M2 was identified with heterozygosity in 3 of the SNPs in the URE at remission. Loss of heterozygosity was further found in this patient at 2 polymorphic sites in the 5' promoter region and in 2 intronic sites flanking exon 4, thus suggesting loss of heterozygosity covering at least 40 kb of the PU.1 locus. Consistently, PU.1 expression in this patient was markedly reduced. Our study suggests that heterozygous deletion of the PU.1 locus can be associated with human AML.

Unfolded protein response suppresses CEBPA by induction of calreticulin in acute myeloid leukaemia.

The unfolded protein response (UPR) is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). The role of the UPR during leukemogenesis is unknown so far. Here, we studied the induction of mediators of the UPR in leukaemic cells of AML patients. Increased expression of the spliced variant of the X-box binding protein 1 (XBP1s) was detected in 17.4% (16 of 92) of AML patients. Consistent with activated UPR, this group also had increased expression of ER-resident chaperones such as the 78 kD glucose-regulated protein (GRP78) and of calreticulin. Conditional expression of calreticulin in leukaemic U937 cells was found to increase calreticulin binding to the CEBPA mRNA thereby efficiently blocking translation of the myeloid key transcription factor CEBPA and ultimately affecting myeloid differentiation. Consequently, leukaemic cells from AML patients with activated UPR and thus increased calreticulin levels showed in fact suppressed CEBPA protein expression. We identified two functional ER stress response elements (ERSE) in the calreticulin promoter. The presence of NFY and ATF6, as well as an intact binding site for YY1 within these ERSE motifs were essential for mediating sensitivity to ER stress and activation of calreticulin. Thus, we propose a model of the UPR being activated in a considerable subset of AML patients through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation.

Lineage-specific transcription factor aberrations in AML.

Transcription factors play a key role in the commitment of hematopoietic stem cells to differentiate into specific lineages [78]. This is particularly important in that a block in terminal differentiation is the key contributing factor in acute leukemias. This general theme of the role of transcription factors in differentiation may also extend to other tissues, both in terms of normal development and cancer. Consistent with the role of transcription factors in hematopoietic lineage commitment is the frequent finding of aberrations in transcription factors in AML patients. Here, we intend to review recent findings on aberrations in lineage-restricted transcription factors as observed in patients with acute myeloid leukemia (AML).

Activation of the unfolded protein response is associated with favorable prognosis in acute myeloid leukemia.

PURPOSE: The unfolded protein response is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum. Previous studies suggest that the unfolded protein response is activated in some cancer cell lines and involved in tumor development. The role of the unfolded protein response during leukemogenesis is unknown thus far. EXPERIMENTAL DESIGN: Here, we assessed the induction of key effectors of the unfolded protein response in leukemic cells at diagnosis of 105 acute myeloid leukemia (AML) patients comprising all subtypes. We determined the formation of the spliced variant of the X-box-binding protein 1 (XBP1) mRNA, as well as expression levels of calreticulin, GRP78, and CHOP mRNA. RESULTS: The formation of the spliced variant of XBP1s was detectable in 16.2% (17 of 105) of AML patients. Consistent with activated unfolded protein response, this group also had significantly increased expression of calreticulin, GRP78, and CHOP. AML patients with activated unfolded protein response had lower WBC counts, lactate dehydrogenase levels, and more frequently, secondary AML. The incidence of fms-related tyrosine kinase 3 (FLT3) mutations was significantly lower in patients with activated unfolded protein response. In addition, an association was observed between activated unfolded protein response and deletion of chromosome 7. Finally, the clinical course of AML patients with activated unfolded protein response was more favorable with lower relapse rate (P = 0.0182) and better overall (P = 0.041) and disease-free survival (P = 0.022). CONCLUSIONS: These results suggest that the unfolded protein response is activated in a considerable subset of AML patients. AML patients with activated unfolded protein response present specific clinical characteristics and a more favorable course of the disease.

Multimorbidity and cardiovascular disease: a perspective on low- and middle-income countries.

New and changing patterns of multimorbidity (MM), i.e., multiple concurrent acute or chronic diseases in a person, are emerging in low- and middle-income countries (LMICs). The interplay of underlying population-specific factors and lifestyle habits combined with the colliding epidemics of communicable and non-communicable diseases presents new disease combinations, complexities and risks that are not common in high-income countries (HICs). The complexities and risks include those arising from potentially harmful drug-drug and drug-disease interactions (DDIs), the management of which may be considered as MM in the true sense. A major concern in LMICs is the increasing burden of leading cardiovascular diseases, prevalence of associated risk factors and co-occurrence with other morbidities. New models of MM management and integrated care can respond to the needs of specific multimorbid populations, with some LMICs making substantial progress (e.g., integration of tuberculosis and HIV services in South Africa). But there is a dearth of relevant data on the changing patterns and underlying factors and determinants of MM, the associated complexities and risks of DDIs in MM management, and the barriers to integrated care in LMICs. This requires careful attention.

Unmet challenges in treating hypertension in patients with borderline personality disorder: A systematic review.

BACKGROUND: Caregivers encounter serious and substantial challenges in managing hypertension in patients with subclinical or clinical borderline personality disorder (BPD). These challenges include therapeutic conflicts resulting from harmful drug-drug, and drug-disease interactions. Current guidelines provide no recommendations for concurrent psychotropic and antihypertensive treatment of hypertensive BPD patients who are at even greater cardiovascular risk. METHODS: We conducted a systematic literature review to assess the extent of available evidence on prevalence rates, cardiovascular risk factors, therapeutic conflicts, and evidence-based treatment recommendations for patients with co-occurring hypertension and BPD. Search terms were combined for hypertension and BPD in PubMed, MEDLINE, EMBASE, Cochrane, and PsycINFO databases. RESULTS: We included 11 articles for full-text evaluation and found a very high prevalence of hypertension and substantial cardiovascular risk in studies on co-occurring BPD and hypertension. However, we identified neither studies on harmful drug-drug and drug-disease interactions nor studies with treatment recommendations for co-occurring hypertension and BPD. CONCLUSIONS: Increased prevalence of hypertension in BPD patients, and therapeutic conflicts of psychotropic agents strongly suggest careful evaluation of treatment strategies in this patient group. However, no studies or guidelines recommend specific therapies or strategies to resolve therapeutic conflicts in patients with hypertension and BPD. This evidence gap needs attention in this population at high risk for cardiovascular disease.

The maximum alcohol withdrawal syndrome score associates with worse clinical outcomes-A retrospective cohort study.

BACKGROUND: The Wetterling alcohol withdrawal syndrome (AWS) scale determines withdrawal severity and guides treatment. We investigated associations between maximum AWS scores and clinical outcomes. METHODS: This retrospective cohort study considered AWS assessments measured from 8/2015-8/2017. We used multivariable linear and logistic regression to analyze associations between the maximum score and increased length of stay (LOS) and in-hospital mortality, respectively. Firstly, we investigated the maximum score of all AWS assessments any time during the stay, secondly, the maximum measured only within the first 3 days of withdrawal. RESULTS: A total of 2,464 hospital stays showed that, patients with "mild" (<6), "moderate" (6-9), and "severe" (>9) maximum scores had median LOS of 5.93, 9.35, 14.71 days, mortality was 1.7%, 4.8%, 8.0%, respectively. Regression showed that a higher maximum score was independently associated with increased LOS and mortality (both p < 0.001). Based on the maximum AWS score within the first 3 days, the median LOS was 6.18, 9.00, 12.89 days, mortality was 2.2%, 3.6%, 7.6%, respectively. A higher maximum score in the first 3 days was independently associated with increased LOS (p = 0.036) and mortality (p = 0.001). Severe maximum AWS scores within 3 days of withdrawal had an odds ratio of 2.53 (95% CI: 1.27, 4.82; p = 0.0060) for in-hospital death. CONCLUSIONS: Maximum AWS scores associate independently with increased LOS and in-hospital mortality. This association is reproducible within the first 3 days of withdrawal. Development of such a 3-day tool could help clinicians assess the risk of worse clinical outcomes early on and adjust care accordingly.

Improved survival rates of AML patients following admission to the intensive care unit.

Induction chemotherapy in AML patients may have life-threatening side effects requiring intensive care unit (ICU) treatment. We analyzed all AML patients receiving intensive chemotherapy at a single academic center between 01/2006-12/2016. At least one ICU admission was observed in 32% (76/240) patients, and 33% of those died following ICU admission. Whereas the ICU admission proportion remained stable, mortality after ICU admission decreased from 14% (2006-2008) to 3% (2014-2016; p = .056). The number of failing organ systems inversely correlated with surviving ICU admission (p < .001). Sepsis and renal, cardiac and pulmonary failure were each associated with higher mortality. With increasing ICU duration, survival probability decreased (p < .001), but remained >50% even after 14 days of ICU treatment. Progression-free and overall survival were comparable between ICU surviving patients and patients never needing ICU support. In conclusion, outcome after ICU admission of AML patients has substantially improved in recent years.

Rebound Thrombocytosis after Induction Chemotherapy is a Strong Biomarker for Favorable Outcome in AML Patients.

Whereas the molecular events underlying acute myeloid leukemia (AML) are increasingly identified, dynamics of hematologic recovery following induction chemotherapy remain mysterious. Platelet recovery may vary between incomplete and excess recovery among patients achieving remission. We analyzed platelet recovery after the first induction cycle in 291 consecutive AML patients. We defined excess platelet rebound (EPR) as platelet increase above 500 G/L. We observed EPR in 120 (41.2%) patients. EPR+ patients had lower platelets at diagnosis, higher marrow infiltration, more frequently NPM1 mutations, and were associated with ELN favorable risk. Absence of EPR correlated with complex karyotypes, ELN intermediate-I and adverse risk, and therapy-related AML. Overall survival was better in EPR+ patients than EPR- (median 125 vs 41 months; p = 0.04), as was disease-free survival. By multivariate analysis, EPR+ was an independent parameter associated with favorable survival. Plasma thrombopoietin (TPO) levels at diagnosis indicated EPR+ (p < 0.0001), while GATA-1, GATA-2, and MPL mRNA expression did not differ between EPR+ and EPR- patients. Finally, transcription factors blocking early megakaryopoiesis were upregulated in EPR- patients, while NFE2 involved in late megakaryocyte differentiation was increased in EPR+ patients. Our work identifies mechanisms involved in platelet recovery after induction chemotherapy.

HIC1 tumour suppressor gene is suppressed in acute myeloid leukaemia and induced during granulocytic differentiation.

A hallmark of acute myeloid leukaemia (AML) is a block in differentiation caused by deregulated gene expression. The tumour suppressor Hypermethylated In Cancer 1 (HIC1) is a transcriptional repressor, which is epigenetically silenced in solid cancers. HIC1 mRNA expression was found to be low in 128 patient samples of AML and CD34+ progenitor cells when compared with terminally differentiated granulocytes. HIC1 mRNA was induced in a patient with t(15;17)-positive acute promyelocytic leukaemia receiving all-trans retinoic acid (ATRA) therapy. We therefore investigated whether HIC1 plays a role in granulocytic differentiation and whether loss of function of this gene might contribute to the differentiation block in AML. We evaluated HIC1 mRNA levels in HL-60 and U-937 cells upon ATRA-induced differentiation and in CD34+ progenitor cells after granulocyte colony-stimulating factor-induced differentiation. In both models of granulocytic differentiation, we observed significant HIC1 induction. When HIC1 mRNA was suppressed in HL-60 cells using stably expressed short hairpin RNA targeting HIC1, granulocytic differentiation was altered as assessed by CD11b expression. Bisulphite sequencing of GC-rich regions (CpG islands) in the HIC1 promoter provided evidence that the observed suppression in HL-60 cells was not because of promoter hypermethylation. Our findings indicate a role for the tumour suppressor gene HIC1 in granulocytic differentiation. Low expression of HIC1 may very well contribute to pathogenic events in leukaemogenesis.

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells.

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.