Hematopoietic stem cell involvement in BCR-ABL1-positive ALL as a potential mechanism of resistance to blinatumomab therapy.

The bispecific T-cell engager blinatumomab targeting CD19 can induce complete remission in relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, some patients ultimately relapse with loss of CD19 antigen on leukemic cells, which has been established as a novel mechanism to escape CD19-specific immunotherapies. Here, we provide evidence that CD19-negative (CD19-) relapse after CD19-directed therapy in BCP-ALL may be a result of the selection of preexisting CD19- malignant progenitor cells. We present 2 BCR-ABL1 fusion-positive BCP-ALL patients with CD19- myeloid lineage relapse after blinatumomab therapy and show BCR-ABL1 positivity in their hematopoietic stem cell (HSC)/progenitor/myeloid compartments at initial diagnosis by fluorescence in situ hybridization after cell sorting. By using the same approach with 25 additional diagnostic samples from patients with BCR-ABL1-positive BCP-ALL, we identified HSC involvement in 40% of the patients. Patients (6 of 8) with major BCR-ABL1 transcript encoding P210BCR-ABL1 mainly showed HSC involvement, whereas in most of the patients (9 of 12) with minor BCR-ABL1 transcript encoding P190BCR-ABL1, only the CD19+ leukemia compartments were BCR-ABL1 positive (P = .02). Our data are of clinical importance, because they indicate that both CD19+ cells and CD19- precursors should be targeted to avoid CD19- relapses in patients with BCR-ABL1-positive ALL.

Differential but direct abolishment of human regulatory T cell suppressive capacity by various TLR2 ligands.

CD4(+)CD25(high) regulatory T cells (Tregs) control cellular immune responses and maintain peripheral tolerance. We investigated whether TLR2 ligands are able to abrogate Treg-induced suppression in humans based on different reports about effects of triacylated lipopeptide Pam(3)CSK4 in mice. Pretreatment of human Tregs with a mixture of TLR2 ligands Pam(2)CSK4, FSL-1, and Pam(3)CSK4 reduced the Treg-mediated suppression of CD4(+)CD25(-) responder T cells in the majority of the analyzed donors. Differential effects of individual TLR2 ligands are explained by usage of different TLR2 heterodimers in the recognition of Pam(2)CSK4, FSL-1, and Pam(3)CSK4. In contrast to the murine system, TLR2 ligand-mediated abrogation of human Treg function was not associated with a downregulation of FoxP3 transcription factor. Furthermore, our results excluded an effect of TLR2 ligands on granzyme A/B release by human Tregs as a potential mechanism to abolish Treg-mediated suppression. Our data suggest that a downregulation of p27(Kip1) and restoration of Akt phosphorylation in human Tregs pretreated with TLR2 ligands result in a reversal of suppression on responder T cells. Moreover, our data indicate that a mixture of TLR2 ligands can be used to modulate human Treg activity.

Interaction with XIAP prevents full caspase-3/-7 activation in proliferating human T lymphocytes.

Caspases are essential mediators of cytokine release and apoptosis. Additionally, caspase activity is required for the proliferation of naive T lymphocytes. It remained unclear how proliferating cells are able to cope with the pro-apoptotic activity especially of effector caspases-3 and -7. Possible reasons might include limited subcellular localization of active caspases or inhibition by endogenous caspase inhibitors. Here, we compared the activation of various caspases in proliferating human T cells with that in apoptotic cells. We show that cleaved caspases-3/-7 appear to be widely distributed in apoptotic cells while they are largely confined to the cytoplasm in proliferating cells. Additionally, in proliferating T cells caspase-3 remains incompletely cleaved, while in apoptotic cells fully mature caspase-3 is generated. We provide evidence that during T cell proliferation the intracellular caspase inhibitor X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspases-3/-7, thereby blocking their full activation, substrate cleavage, and cell death. The lack of substrate cleavage might also lead to the observed limited subcellular distribution of caspases-3/-7. After induction of apoptosis, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/DIABLO) is released from mitochondria, resulting in the abrogation of the inhibitory effect of XIAP, full activation of caspases-3/-7, and apoptosis.

The Fas-associated death domain protein/caspase-8/c-FLIP signaling pathway is involved in TNF-induced activation of ERK.

The cytokine TNF activates multiple signaling pathways leading to cellular responses ranging from proliferation and survival to apoptosis. While most of these pathways have been elucidated in detail over the past few years, the molecular mechanism leading to the activation of the MAP kinases ERK remains ill defined and is controversially discussed. Therefore, we have analyzed TNF-induced ERK activation in various human and murine cell lines and show that it occurs in a cell-type-specific manner. In addition, we provide evidence for the involvement of the signaling components Fas-associated death domain protein (FADD), caspase-8, and c-FLIP in the pathway activating ERK in response to TNF. This conclusion is based on the following observations: (I) Overexpression of FADD, caspase-8, or a c-FLIP protein containing the death effector domains only leads to enhanced and prolonged ERK activation after TNF treatment. (II) TNF-induced ERK activation is strongly diminished in the absence of FADD. Interestingly, the enzymatic function of caspase-8 is not required for TNF-induced ERK activation. Additional evidence suggests a role for this pathway in the proliferative response of murine fibroblasts to TNF.

Caspase inhibition blocks human T cell proliferation by suppressing appropriate regulation of IL-2, CD25, and cell cycle-associated proteins.

Caspases have been described as proteases essential for the release of certain cytokines and for initiation as well as execution of apoptosis. Increasing evidence indicates, however, that caspase activity is also required for activation-induced proliferation of mature T lymphocytes. The molecular mechanism, how caspase activity facilitates T cell proliferation, is still controversially discussed. In this study, we show that proliferation of human T cells in response to a specific antigenic stimulus is completely prevented by caspase inhibition. In addition, we demonstrate that this lack of proliferation is due to a failure to initiate cell cycle progression, but not the result of increased T cell death. Our results demonstrate that caspase inhibition leads to strongly reduced IL-2 release, failure to up-regulate CD25, and a lack of proper regulation of cell cycle-associated proteins. Furthermore, T cell proliferation was partially rescued by addition of exogenous IL-2. Using Jurkat cells, we show that in the absence of caspase-8, the mitogen-induced activation of the transcription factor NF-kappaB is moderately diminished, while the activity of the composite element CD28 response element and NF-IL-2B AP-1 sites is strongly reduced. Finally, we provide evidence that caspase inhibition suppresses the activation of purified monocytes by bacterial Ags.

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-kappaB, elicits caspase activity, and enhances cytotoxicity.

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.

Species-specific differences in the usage of several caspase substrates.

The activation of caspases cleaving a plethora of specific substrates is pivotal for initiation as well as execution of apoptosis. The recognition motif for caspases is a tetrapeptide sequence containing an essential aspartic acid residue at the fourth position (often DXXD). Here, we report that the caspase cleavage sites of most identified substrates show a high degree of conservation between different species. However, we have identified differences in the cleavage sites of five substrates between murine and human proteins leading to either select processing in only one species or to different cleavage patterns. Finally, we provide evidence that murine c-Abl but not its human homolog serves as efficient substrate during apoptosis.

Upregulation of p21(WAF1/Cip1) precedes tumor necrosis factor-induced necrosis-like cell death.

The molecular mechanisms mediating death receptor-induced caspase-independent necrotic cell death are still largely unknown. We have previously reported that NIH3T3 cells are sensitized by caspase inhibition to death receptor-induced cytotoxicity leading to a necrosis-like cell death. In addition, we have identified an important role of cell cycle progression for this sensitization effect. Here, we report that tumor necrosis factor-induced necrotic death is preceded by an upregulation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Increased expression of p21(WAF1/Cip1) occurs prior to cell death in the nucleus, where it binds to a cyclin-dependent kinase indicating its functionality. The use of specific pharmacological inhibitors revealed a partial involvement of p38 mitogen-activated protein kinase in the upregulation of p21(WAF1/Cip1). Inhibition of p21(WAF1/Cip1) upregulation prevents a previously observed delay of the cells in the G2/M phase of the cell cycle thereby augmenting, not inhibiting cell death.