Christian R. Geest

MAPK signaling pathways in the regulation of hematopoiesis

The MAPKs are a family of serine/threonine kinases that play an essential role in connecting cell‐surface receptors to changes in transcriptional programs. MAPKs are part of a three‐component kinase module consisting of a MAPK, an upstream MEK, and a MEKK that couples the signals from cell‐surface receptors to trigger downstream pathways. Three major groups of MAPKs have been characterized in mammals, including ERKs, JNKs, and p38MAPKs. Over the last decade, extensive work has established that these proteins play critical roles in the regulation of a wide variety of cellular processes including cell growth, migration, proliferation, differentiation, and survival. It has been demonstrated that ERK, JNK, and p38MAPK activity can be regulated in response to a plethora of hematopoietic cytokines and growth factors that play critical roles in hematopoiesis. In this review, we summarize the current understanding of MAPK function in the regulation of hematopoiesis in general and myelopoiesis in particular. In addition, the consequences of aberrant MAPK activation in the pathogenesis of various myeloid malignancies will be discussed.

Tipping the Noxa/Mcl-1 Balance Overcomes ABT-737 Resistance in Chronic Lymphocytic Leukemia

Purpose: Chronic lymphocytic leukemia (CLL) cells in lymph nodes (LN), from which relapses are postulated to originate, display an antiapoptotic profile in contrast to CLL cells from peripheral blood (PB). The BH3 mimetic ABT-737 antagonizes the antiapoptotic proteins Bcl-XL and Bcl-2 but not Mcl-1 or Bfl-1. Previously, it was shown that CD40-stimulated CLL cells were resistant to ABT-737. We aimed to define which antiapoptotic proteins determine resistance to ABT-737 in CLL and whether combination of known antileukemia drugs and ABT-737 was able to induce apoptosis of CD40-stimulated CLL cells. Experimental Design: To mimic the LN microenvironment, PB lymphocytes of CLL patients were cultured on feeder cells expressing CD40L and treated with ABT-737 with or without various drugs. In addition, we carried out overexpression or knockdown of pro- and antiapoptotic proteins in immortalized primary B cells. Results: Upon CD40 stimulation patient-specific variations in ABT-737 sensitivity correlated with differences in levels of Mcl-1 and its antagonist Noxa. Knockdown of Noxa, as well as Mcl-1 overexpression, corroborated the importance of the Noxa/Mcl-1 ratio in determining the response to ABT-737. Inhibition of NF-κB resulted in increased Noxa levels and enhanced sensitivity to ABT-737. Interestingly, increasing the Noxa/Mcl-1 ratio, by decreasing Mcl-1 (dasatinib and roscovitine) or increasing Noxa levels (fludarabine and bortezomib), resulted in synergy with ABT-737. Conclusions: Thus, the Noxa/Mcl-1 balance determines sensitivity to ABT-737 in CD40-stimulated CLL cells. These data provide a rationale to investigate the combination of drugs which enhance the Noxa/Mcl-1 balance with ABT-737 to eradicate CLL in chemoresistant niches. Clin Cancer Res; 18(2); 487–98. ©2011 AACR.

Differential expression of leukocyte‐associated Ig‐like receptor‐1 during neutrophil differentiation and activation

Inhibitory receptors containing immunoreceptor tyrosine‐based inhibitory motifs play an important regulatory role in immune cell activation. In addition, several studies suggest that these receptors are involved in the regulation of hematopoietic cell differentiation. Here, we have investigated the expression of leukocyte‐associated immunoglobulin‐like receptor‐1 (LAIR‐1), an inhibitory receptor expressed on most peripheral blood leukocytes and on CD34+ hematopoietic progenitor cells, in neutrophil differentiation and activation. We found that although LAIR‐1 was expressed on peripheral blood eosinophils, cell‐surface expression on mature neutrophils was low, suggesting that LAIR‐1 expression is regulated during granulocyte differentiation. Indeed, the promyeloid cell line HL‐60 expressed LAIR‐1, but the expression decreased during chemical‐induced differentiation toward neutrophils. Similarly, in bone marrow‐derived neutrophil precursors, the most immature cells expressed LAIR‐1, and loss of LAIR‐1 expression was associated with neutrophil maturation. LAIR‐1 was re‐expressed rapidly on the membrane of mature neutrophils upon stimulation with tumor necrosis factor α, granulocyte macrophage‐colony stimulating factor, or N‐formyl‐methionyl‐leucyl‐phenylalanine, indicating that LAIR‐1 may also regulate neutrophil effector function. Our studies suggest that LAIR‐1 may play a regulatory role in differentiation and function of human granulocytes.

Tight control of MEK-ERK activation is essential in regulating proliferation, survival, and cytokine production of CD34(+)-derived neutrophil progenitors

A plethora of extracellular stimuli regulate growth, survival, and differentiation responses through activation of the MEK-ERK MAPK signaling module. Using CD34+ hematopoietic progenitor cells, we describe a novel role for the MEK-ERK signaling module in the regulation of proliferation, survival, and cytokine production during neutrophil differentiation. Addition of the specific MEK1/2 inhibitor U0126 resulted in decreased proliferation of neutrophil progenitors. Conversely, transient activation of a conditionally active MEK1 mutant resulted in the expansion of progenitor cells, which thereafter differentiated normally into mature neutrophils. In contrast, chronic MEK1 activation was found to induce cell death of CD34+ neutrophil progenitors. Microarray analysis of CD34+ progenitor cells showed that activation of MEK1 resulted in changes in expression of a variety of cell-cycle modulating genes. Furthermore, conditional activation of MEK1 resulted in a dramatic increase in the expression of mRNA transcripts encoding a large number of hematopoietic cytokines, chemokines, and growth factors. These findings identify a novel role for MEK-ERK signaling in regulating the balance between proliferation and apoptosis during neutrophil differentiation, and they suggest the need for tight control of MEK-ERK activation to prevent the development of bone marrow failure.

p38 MAP Kinase Inhibits Neutrophil Development Through Phosphorylation of C/EBPα on Serine 21†‡

Many extracellular stimuli regulate growth, survival, and differentiation responses through activation of the dual specificity mitogen activated protein kinase (MAPK) kinase three (MKK3) and its downstream effector p38 MAPK. Using CD34+ hematopoietic progenitor cells, here we describe a novel role for MKK3‐p38MAPK in the regulation of myelopoiesis. Inhibition of p38MAPK utilizing the pharmacological inhibitor SB203580, enhanced neutrophil development ex vivo, but conversely reduced eosinophil differentiation. In contrast, constitutive activation of MKK3 dramatically inhibited neutrophil differentiation. Transplantation of β2‐microglobulin−/− nonobese diabetic/severe combined immune deficient (NOD/SCID) mice with CD34+ cells ectopically expressing constitutively active MKK3 resulted in reduced neutrophil differentiation in vivo, whereas eosinophil development was enhanced. Inhibitory phosphorylation of CCAAT/enhancer binding protein α (C/EBPα) on serine 21 was induced upon activation of p38MAPK. Moreover, ectopic expression of a non‐phosphorylatable C/EBPα mutant was sufficient to abrogate MKK3‐induced inhibition of neutrophil development. Furthermore, treatment of CD34+ progenitors from patients with severe congenital neutropenia with SB203580 restored neutrophil development. These results establish a novel role for MKK3‐p38MAPK in the regulation of lineage choices during myelopoiesis through modulation of C/EBPα activity. This signaling module may thus provide an important therapeutic target in the treatment of bone marrow failure. STEM CELLS 2009;27:2271–2282

Mammalian target of rapamycin activity is required for expansion of CD34(+) hematopoietic progenitor cells

The protein kinase mammalian target of rapamycin (mTOR) plays an essential role in the control of protein synthesis. In this paper, Geest and colleagues show that mTOR inhibition by rapamycin reduces the expansion of committed myeloid progenitors, but leaves the more primitive hematopoietic compartment unaffected. Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic syndromes, suggesting that correct regulation of mammalian target of rapamycin is critical for normal hematopoiesis. Design and Methods An ex vivo granulocyte differentiation system was utilized to investigate the role of mammalian target of rapamycin in the regulation of myelopoiesis. Results Inhibition of mammalian target of rapamycin activity, with the pharmacological inhibitor rapamycin, dramatically reduced hematopoietic progenitor expansion, without altering levels of apoptosis or maturation. Moreover, analysis of distinct hematopoietic progenitor populations revealed that rapamycin treatment inhibited the expansion potential of committed CD34+ lineage-positive progenitors, but did not affect early hematopoietic progenitors. Further examinations showed that these effects of rapamycin on progenitor expansion might involve differential regulation of protein kinase B and mammalian target of rapamycin signaling. Conclusions Together, these results indicate that mammalian target of rapamycin activity is essential for expansion of CD34+ hematopoietic progenitor cells during myelopoiesis. Modulation of the mammalian target of rapamycin pathway may be of benefit in the design of new therapies to control hematologic malignancies.