Early-phenotype CAR-T cells for the treatment of pediatric cancers.

Chimeric antigen receptor (CAR)-T-cell therapy is a promising approach for the treatment of childhood cancers, particularly high-risk tumors that fail to respond to standard therapies. CAR-T cells have been highly successful in treating some types of hematological malignancies. However, CAR-T cells targeting solid cancers have had limited success so far for multiple reasons, including their poor long-term persistence and proliferation. Evidence is emerging to show that maintaining CAR-T cells in an early, less-differentiated state in vitro results in superior persistence, proliferation, and antitumor effects in vivo. Children are ideal candidates for receiving less-differentiated CAR-T cells, because their peripheral T-cell pool primarily comprises naïve cells that could readily be harvested in large numbers to generate early-phenotype CAR-T cells. Although several studies have reported different approaches to successfully generate early CAR-T cells, there are only a few clinical trials testing these in adult patients. No trials are currently testing early CAR-T cells in children. Here, we summarize the different strategies used to maintain CAR-T cells in an early phenotypic stage and present evidence suggesting that this approach may be particularly relevant to treating childhood cancers.

In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8.

BACKGROUND: Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. METHODS: A method is described to massively expand bone marrow-derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. RESULTS: IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). CONCLUSION: A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.

DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9.

MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. Here, we show that after UV radiation, MIHA prevented apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functioned after release of cytochrome c and DIABLO from the mitochondria and was able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.

Identification of mammalian mitochondrial proteins that interact with IAPs via N-terminal IAP binding motifs.

Direct IAP binding protein with low pI/second mitochondrial activator of caspases, HtrA2/Omi and GstPT/eRF3 are mammalian proteins that bind via N-terminal inhibitor of apoptosis protein (IAP) binding motifs (IBMs) to the baculoviral IAP repeat (BIR) domains of IAPs. These interactions can prevent IAPs from inhibiting caspases, or displace active caspases, thereby promoting cell death. We have identified several additional potential IAP antagonists, including glutamate dehydrogenase (GdH), Nipsnap 3 and 4, CLPX, leucine-rich pentatricopeptide repeat motif-containing protein and 3-hydroxyisobutyrate dehydrogenase. All are mitochondrial proteins from which N-terminal import sequences are removed generating N-terminal IBMs. Whereas most of these proteins have alanine at the N-terminal position, as observed for previously described antagonists, GdH has an N-terminal serine residue that is essential for X-linked IAP (XIAP) interaction. These newly described IAP binding proteins interact with XIAP mainly via BIR2, with binding eliminated or significantly reduced by a single point mutation (D214S) within this domain. Through this interaction, many are able to antagonise XIAP inhibition of caspase 3 in vitro.

Enhancing venetoclax activity in acute myeloid leukemia by co-targeting MCL1.

Targeted therapies are frequently combined with standard cytotoxic drugs to enhance clinical response. Targeting the B-cell lymphoma 2 (BCL-2) family of proteins is an attractive option to combat chemoresistance in leukemia. Preclinical and clinical studies indicate modest single-agent activity with selective BCL-2 inhibitors (for example, venetoclax). We show that venetoclax synergizes with cytarabine and idarubicin to increase antileukemic efficacy in a TP53-dependent manner. Although TP53 deficiency impaired sensitivity to combined venetoclax and chemotherapy, higher-dose idarubicin was able to suppress MCL1 and induce cell death independently of TP53. Consistent with an MCL1-specific effect, cell death from high-dose idarubicin was dependent on pro-apoptotic Bak. Combining higher-dose idarubicin with venetoclax was able to partially overcome resistance in Bak-deficient cells. Using inducible vectors and venetoclax to differentially target anti-apoptotic BCL-2 family members, BCL-2 and MCL1 emerged as critical and complementary proteins regulating cell survival in acute myeloid leukemia. Dual targeting of BCL-2 and MCL1, but not either alone, prolonged survival of leukemia-bearing mice. In conclusion, our findings support the further investigation of venetoclax in combination with standard chemotherapy, including intensified doses of idarubicin. Venetoclax should also be investigated in combination with direct inhibitors of MCL1 as a chemotherapy-free approach in the future.

Functionally distinct roles for different miR-155 expression levels through contrasting effects on gene expression, in acute myeloid leukaemia.

Enforced expression of microRNA-155 (miR-155) in myeloid cells has been shown to have both oncogenic or tumour-suppressor functions in acute myeloid leukaemia (AML). We sought to resolve these contrasting effects of miR-155 overexpression using murine models of AML and human paediatric AML data sets. We show that the highest miR-155 expression levels inhibited proliferation in murine AML models. Over time, enforced miR-155 expression in AML in vitro and in vivo, however, favours selection of intermediate miR-155 expression levels that results in increased tumour burden in mice, without accelerating the onset of disease. Strikingly, we show that intermediate and high miR-155 expression also regulate very different subsets of miR-155 targets and have contrasting downstream effects on the transcriptional environments of AML cells, including genes involved in haematopoiesis and leukaemia. Furthermore, we show that elevated miR-155 expression detected in paediatric AML correlates with intermediate and not high miR-155 expression identified in our experimental models. These findings collectively describe a novel dose-dependent role for miR-155 in the regulation of AML, which may have important therapeutic implications.

Nerve growth factor signaling through p75 induces apoptosis in Schwann cells via a Bcl-2-independent pathway.

Apoptosis is involved in the regulation of Schwann cell numbers during normal development and after axonal damage, but the molecular regulation of Schwann cell death remains unknown. We have used stably transfected rat Schwann cell lines to study the potential roles of nerve growth factor (NGF), the antiapoptotic protein Bcl-2 and the cytokine response modifier A (CrmA) in modulating Schwann cell death in vitro. Bcl-2 inhibited Schwann cell apoptosis induced by survival factor withdrawal, whereas CrmA did not. In contrast, Bcl-2-transfected Schwann cells were susceptible to apoptosis in response to exogenous NGF, whereas CrmA-expressing cell lines were resistant. Demonstration of high levels of the low-affinity neurotrophin receptor p75 but not the high-affinity TrkA receptor on the Bcl-2-transfected cell lines suggested that the NGF-induced killing was mediated by p75. This was confirmed by resistance of Schwann cells isolated from p75 knockout mice to the NGF-induced cell death. Nerve growth factor also promoted the death of wild-type mouse and rat Schwann cells in the absence of survival factor withdrawal. Endogenous Bcl-2 mRNA was expressed by wild-type Schwann cells in all conditions that promoted survival but was downregulated to undetectable levels after survival factor withdrawal. In conclusion, our results demonstrate the existence of two separate pathways that expedite apoptosis in Schwann cells: a Bcl-2-blockable pathway initiated on loss of trophic support, and a Bcl-2-independent, CrmA-blockable pathway mediated via the p75 receptor.

Caspase inhibitors.

Caspases are the key effector molecules of the physiological death process known as apoptosis, although some are involved in activation of cytokines, rather than cell death. They exist in most of our cells as inactive precursors (zymogens) that kill the cell once activated. Caspases can be controlled in two ways. The processing and activation of a caspase can be regulated by molecules such as FADD, APAF-1, Bcl-2 family members, FLIP and IAPs. Active caspases can be controlled by a variety of inhibitors that directly interact with the protease. This review describes the later direct caspase inhibitors that have been identified, products of both viral and cellular genes, and artificial caspase inhibitors that have been developed both as research tools and as pharmaceutical agents to inhibit cell death in vivo.

Anti-apoptotic potential of insect cellular and viral IAPs in mammalian cells.

IAPs were identified as baculoviral proteins that could inhibit the apoptotic response of insect cells to infection. Of the viral IAPs, OpIAP and CpIAP can inhibit apoptosis, whereas AcIAP cannot. OpIAP and some mammalian homologues can inhibit mammalian cell death. Two mammalian IAPs bind to TNFRII associated factors (TRAFs), but the significance of this is unclear. Here we show that Drosophila cellular IAPs and two baculoviral IAPs (OpIAP and CpIAP) can inhibit mammalian cell death induced by overexpression of Caspases 1 and 2. IAPs must act on conserved components of the apoptotic mechanism, but as none of these IAPs could bind TRAF proteins, TRAFs are not likely to be important for IAP mediated apoptosis inhibition. As OpIAP protected against death induced by ligation of TNF receptor family members, but not by factor nor serum withdrawal from dependent cells, it can inhibit certain apoptotic pathways without affecting others.

The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis.

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the P1 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide-substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.

The Caenorhabditis elegans CED-9 protein does not directly inhibit the caspase CED-3, in vitro nor in yeast.

A genetically defined pathway orchestrates the removal of 131 of the 1090 somatic cells generated during the development of the hermaphrodite nematode Caenorhabditis elegans. Regulation of apoptosis is highly evolutionarily conserved and the nematode cell death pathway is a valuable model for studying mammalian apoptotic pathways, the dysregulation of which can contribute to numerous diseases. The nematode caspase CED-3 is ultimately responsible for the destruction of worm cells in response to apoptotic signals, but it must first be activated by CED-4. CED-9 inhibits programmed cell death and considerable data have demonstrated that CED-9 can directly bind and inhibit CED-4. However, it has been suggested that CED-9 may also directly inhibit CED-3. In this study, we used a yeast-based system and biochemical approaches to explore this second potential mechanism of action. While we confirmed the ability of CED-9 to inhibit CED-4, our data argue that CED-9 can not directly inhibit CED-3.

Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is dependent on both Apaf-1 and caspase-9.

We have generated rat monoclonal antibodies that specifically recognise caspase-2 from many species, including mouse, rat and humans. Using these antibodies, we have investigated caspase-2 expression, subcellular localisation and processing. We demonstrate that caspase-2 is expressed in most tissues and cell types. Cell fractionation and immunohistochemistry experiments show that caspase-2 is found in the nuclear and cytosolic fractions, including a significant portion present in the Golgi complex. We found that caspase-2 is processed in response to many apoptotic stimuli but experiments with caspase-2 deficient mice demonstrated that it is not required for apoptosis of thymocytes or dorsal root ganglia (DRG) neurons in response to a variety of cytotoxic stimuli. Caspase-2 processing does not occur in thymocytes lacking Apaf-1 or caspase-9, suggesting that in this cell type, activation of caspase-2 occurs downstream of apoptosome formation.

Dexamethasone prevents apoptosis in a neonatal rat model of hypoxic-ischemic encephalopathy (HIE) by a reactive oxygen species-independent mechanism.

It has previously been shown, in a neonatal rat model of hypoxic-ischemic encephalopathy (HIE), that neuronal injury can be attenuated by pretreatment with dexamethasone. The mechanism by which dexamethasone exerts this protective effect is not known. Using the same neonatal rat model of HIE, we found pretreatment with dexamethasone to have no effect on the generation of superoxide radical, products of lipid peroxidation, peroxynitrite-mediated tissue damage or bcl-2 protein expression. However, dexamethasone did inhibit the induction of c-fos transcription seen following HIE, and subsequent evidence of apoptosis. We conclude that it is possible to limit hypoxic-ischemic neuronal injury, despite the continued production of reactive oxygen species, by interventions which block the cascade of events culminating in apoptosis. The involvement of apoptosis in the neuronal injury of HIE, if confirmed in acutely asphyxiated human infants, suggests that there may be a post-injury 'window of opportunity' for neuroprotective interventions.

Epithelial bridging on burn patients following primary excision and grafting.

Early tangential excision and primary grafting of full thickness and deep partial thickness burns has become a widely accepted practice, however, little attention has been paid to the epithelial bridging phenomenon, which occurs most frequently at the edge of these wounds. Over 5 years, 923 burn patients were treated and 407 were grafted, of whom 13 developed sufficiently severe epithelial bridging to require deroofing under general anaesthetic. Histological sections show that the bridges are derived from hair follicles. We advocate that patients with epithelial bridges should be observed in the outpatient department for many weeks and surgical intervention should only be used when they fail to resolve spontaneously.

[Hepatic cholesterol ester storage disease. Two new cases diagnosed in adults]. / Surcharge hépatique en esters de cholestérol. Deux nouveaux cas diagnostiqués chez l'adulte.

Cholesterol ester storage disease is a rare disorder characterized by an hereditary deficiency of lysosomal acid lipase that induces an accumulation of cholesterol ester in most tissues of the body, particularly in liver. The diagnosis is usually made during childhood. The aim of this article is to report two new cases diagnosed in adult age. Two patients, 25 and 20 years old, respectively, presented with hepatomegaly, a slight to moderate increase in serum transaminases, and esophageal varices. In both cases, diagnosis was based on the presence of hypercholesterolemia, fatty infiltration of the liver with lipid droplets in hepatic parenchymal cells, foamy macrophages, hepatic storage of cholesterol esters, and low activity of lysosomal acid lipase. Histological abnormalities were associated with portal and periportal fibrosis in one patient and a micronodular cirrhosis in the other; these lesions were probably the cause of portal hypertension. Fibrosis of varied degrees has been previously reported in cholesterol ester storage disease. Its mechanism remains unclear.

[Iterative intestinal intussusception and appendiceal mucocele in an infant with mucoviscidosis]. / Invaginations intestinales itératives et mucocèle appendiculaire chez un enfant atteint de mucoviscidose.

UNLABELLED: Cystic fibrosis is a common and potentially life-threatening hereditary disease which can affect numerous organs, particularly the digestive tract. CASE REPORT: A 4.5-year-old boy exhibited two little known clinical manifestations: an appendiceal mucocele and repeated intussusceptions. In spite of an appendectomy, intussusception relapsed and an ileocolic resection was necessary 2 years later. DISCUSSION: Appendiceal diseases in cystic fibrosis represent a large spectrum, ie, distention on the appendiceal lumen, engorged with sticky mucous matter, which becomes an appendiceal mucocele, peritonitis with an appendiceal perforation due to delayed diagnosis since acute appendicitis is difficult to diagnose in these patients. Intussusception is rarely observed in cystic fibrosis. CONCLUSION: Appendiceal mucocele could be a cause of intussusception. If an appendectomy is performed, resection of a part of the cecum, around the appendix, could be useful in preventing again mucocele formation.

ER stress does not cause upregulation and activation of caspase-2 to initiate apoptosis.

A recent report claimed that endoplasmic reticulum (ER) stress activates the ER trans-membrane receptor IRE1α, leading to increased caspase-2 levels via degradation of microRNAs, and consequently induction of apoptosis. This observation casts caspase-2 into a central role in the apoptosis triggered by ER stress. We have used multiple cell types from caspase-2-deficient mice to test this hypothesis but failed to find significant impact of loss of caspase-2 on ER-stress-induced apoptosis. Moreover, we did not observe increased expression of caspase-2 protein in response to ER stress. Our data strongly argue against a critical role for caspase-2 in ER-stress-induced apoptosis.

Loss of Prkar1a leads to Bcl-2 family protein induction and cachexia in mice.

Loss of function mutations in the Prkar1a gene are the cause of most cases of Carney complex disorder. Defects in Prkar1a are thought to cause hyper-activation of PKA signalling, which drives neoplastic transformation, and Prkar1a is therefore considered to be a tumour suppressor. Here we show that loss of Prkar1a in genetically modified mice caused transcriptional activation of several proapoptotic Bcl-2 family members and thereby caused cell death. Interestingly, combined loss of Bim and Prkar1a increased colony formation of fibroblasts in culture and promoted their growth as tumours in immune-deficient mice. Apart from inducing apoptosis, systemic deletion of Prkar1a caused cachexia with muscle loss, macrophage activation and increased lipolysis as well as serum triglyceride levels. Loss of single allele of Prkar1a did not enhance tumour development in a skin cancer model, but surprisingly, when combined with the loss of Bim, caused a significant delay in tumorigenesis and this was associated with upregulation of other BH3-only proteins, PUMA and NOXA. These results show that loss of Prkar1a can only promote tumorigenesis when Prkar1a-mediated apoptosis is somehow countered.

Akt1 is the principal Akt isoform regulating apoptosis in limiting cytokine concentrations.

The activation of the Akt signalling in response to cytokine receptor signalling promotes protein synthesis, cellular growth and proliferation. To determine the role of Akt in interleukin-3 (IL-3) signalling, we generated IL-3-dependent myeloid cell lines from mice lacking Akt1, Akt2 or Akt3. Akt1 deletion resulted in accelerated apoptosis at low concentrations of IL-3. Expression of constitutively active Akt1 was sufficient to delay apoptosis in response to IL-3 withdrawal, but not sufficient to induce proliferation in the absence of IL-3. Akt1 prolonged survival of Bim- or Bad-deficient cells, but not cells lacking Puma, indicating that Akt1-dependent repression of apoptosis was in part dependent on Puma and independent of Bim or Bad. Our data show that a key role of Akt1 during IL-3 signalling is to repress p53-dependent apoptosis pathways, including transcriptional upregulation of Puma. Moreover, our data indicate that regulation of BH3-only proteins by Akt is dispensable for Akt-dependent cell survival.