Lentivirus-Mediated BCL-XL Overexpression Inhibits Stem Cell Apoptosis during Ex Vivo Expansion and Provides Competitive Advantage Following Xenotransplantation.

Hematopoietic reconstitution after hematopoietic stem cell transplantation (HSCT) is influenced by the number of transplanted cells. However, under certain conditions donor cell counts are limited and impair clinical outcome. Hematopoietic stem and progenitor cell (HSPC) expansion prior to HSCT is a widely used method to achieve higher donor cell counts and minimize transplantation-related risks such as graft failure or delayed engraftment. Still, expansion in a non-physiological environment can trigger cell death mechanisms and hence counteract the desired effect. We have shown earlier that during HSCT a relevant amount of HSPCs were lost due to apoptosis and that cell death inhibition in donor HSPCs improved engraftment in xenotransplantation experiments. Here, we assessed the effect of combined ex vivo expansion and cell death inhibition on HSPC yield and their reconstitution potential in vivo. During expansion with cytokines and the small molecule inhibitor StemRegenin 1, concomitant lentiviral overexpression of antiapoptotic BCL-XL resulted in an increased yield of transduced HSPCs. Importantly, BCL-XL overexpression enhanced the reconstitution potential of HSPCs in xenotransplantation experiments in vivo. In contrast, treatment with caspase and necroptosis inhibitors had no favorable effects on HSPC yields nor on cell viability. We postulate that overexpression of antiapoptotic BCL-XL, both during ex vivo expansion and transplantation, is a promising approach to improve the outcome of HSCT in situations with limited donor cell numbers. However, such apoptosis inhibition needs to be transient to avoid long-term sequelae like leukemia.

BH3 mimetics and azacitidine show synergistic effects on juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is an aggressive hematopoietic disorder of infancy and early childhood driven by constitutively active RAS signaling and characterized by abnormal proliferation of the granulocytic-monocytic blood cell lineage. Most JMML patients require hematopoietic stem cell transplantation for cure, but the risk of relapse is high for some JMML subtypes. Azacitidine was shown to effectively reduce leukemic burden in a subset of JMML patients. However, variable response rates to azacitidine and the risk of drug resistance highlight the need for novel therapeutic approaches. Since RAS signaling is known to interfere with the intrinsic apoptosis pathway, we combined various BH3 mimetic drugs with azacitidine in our previously established patient-derived xenograft model. We demonstrate that JMML cells require both MCL-1 and BCL-XL for survival, and that these proteins can be effectively targeted by azacitidine and BH3 mimetic combination treatment. In vivo azacitidine acts via downregulation of antiapoptotic MCL-1 and upregulation of proapoptotic BH3-only. The combination of azacitidine with BCL-XL inhibition was superior to BCL-2 inhibition in eliminating JMML cells. Our findings emphasize the need to develop clinically applicable MCL-1 or BCL-XL inhibitors in order to enable novel combination therapies in JMML refractory to standard therapy.

Neonatal Platelets: Lower G12/13 Expression Contributes to Reduced Secretion of Dense Granules.

Despite fully functional primary hemostasis, platelets of healthy neonates exhibit hypoaggregability and secretion defects, which may be adaptations to specific requirements in this developmental stage. The etiologies for reduced signal transduction vary with the type of agonist. The discovered peculiarities are lower receptor densities, reduced calcium mobilization, and functional impairments of G proteins. Reduced secretion of dense granules has been attributed to lower numbers of granules. Signaling studies with adult platelets have shown a regulating effect of the G12/13 signaling pathway on dense granule secretion via RhoA. We comparatively analyzed secretion profiles using flow cytometry and expression levels of Gq, Gi, and G12/13 using Western blot analysis in platelets from cord blood and adults. Furthermore, we evaluated Rho activation after in vitro platelet stimulation with thrombin using a pulldown assay. We observed a markedly reduced expression of the dense granule marker CD63 on neonatal platelets after thrombin stimulation. Gα12/13 expression was significantly decreased in neonatal platelets and correlated with lower Rho activation after thrombin stimulation. We conclude that lower expression of G12/13 in neonatal platelets results in attenuated activation of Rho and may contribute to reduced secretion of dense granules after exposure to thrombin.

No time to die? Intrinsic apoptosis signaling in hematopoietic stem and progenitor cells and therapeutic implications.

PURPOSE OF REVIEW: Dysregulated apoptosis contributes to the pathogenesis of many hematologic malignancies. BH3-mimetics, antagonists of antiapoptotic BCL-2 proteins, represent novel, and promising cancer drugs. While the acute myelosuppressive effects of Venetoclax, the first Food and Drug Administration approved BCL-2 inhibitor, are fairly well described, little is known about side effects of novel BH3-mimetics and effects of chronic Venetoclax treatment. RECENT FINDINGS: Highly relevant publications focused on the effects of acute and chronic Venetoclax therapy, with focus on cell-type specific adaptive mechanisms, the emergence of clonal hematopoiesis, and the selection of BAX-mutated hematopoietic cells in patients treated with Venetoclax for a long period. Important advances were made in understanding primary and secondary Venetoclax resistance and prediction of Venetoclax response. Combination therapies of BH3-mimetics targeting different BCL-2 proteins are highly anticipated. However, human stem and progenitors require both MCL-1 and BCL-XL for survival, and serious myelosuppressive effects of combined MCL-1/BCL-XL inhibition can be expected. SUMMARY: Long-term studies are indispensable to profile the chronic side effects of Venetoclax and novel BH3-mimetics and better balance their risk vs. benefit in cancer therapy. Combination therapies will be powerful, but potentially limited by severe myelosuppression. For precision medicine, a better knowledge of BCL-2 proteins in the healthy and diseased hematopoietic system is required.

Inhibition of the anti-apoptotic protein MCL-1 severely suppresses human hematopoiesis

BH3-mimetics inhibiting anti-apoptotic BCL-2 proteins represent a novel and promising class of antitumor drugs. While the BCL-2 inhibitor venetoclax is already FDA-approved, BCL-XL and MCL-1 inhibitors are currently in early clinical trials. To predict side effects of therapeutic MCL-1 inhibition on the human hematopoietic system, we used RNAi and the small molecule inhibitor S63845 on cord blood-derived CD34+ cells. Both approaches resulted in almost complete depletion of human hematopoietic stem and progenitor cells. As a consequence, maturation into the different hematopoietic lineages was severely restricted and CD34+ cells expressing MCL-1 shRNA showed a very limited engraftment potential upon xenotransplantation. In contrast, mature blood cells survived normally in the absence of MCL-1. Combined inhibition of MCL-1 and BCL-XL resulted in synergistic effects with relevant loss of colony-forming HSPCs already at inhibitor concentrations of 0.1 µM each, indicating "synthetic lethality" of the two BH3-mimetics in the hematopoietic system.

BCL-XL expression is essential for human erythropoiesis and engraftment of hematopoietic stem cells.

The anti-apoptotic BCL-2 proteins (BCL-2, BCL-XL, MCL-1, A1, BCL-W) counteract apoptotic signals emerging during development and under stress conditions, and are thus essential for the survival of every cell. While the "BCL-2 addiction" of different cell types is well described in mouse models, there is only limited information available on the role of different anti-apoptotic BCL-2 proteins in a given human cell type. Here we characterize the role of BCL-XL for survival and function of human hematopoietic cells, with the aim to predict hematological side effects of novel BCL-XL-inhibiting BH3-mimetics and to identify hematological malignancies potentially responsive to such inhibitors. Earlier clinical studies have shown that the combined BCL-2/BCL-XL/BCL-W inhibitor, Navitoclax (ABT-263) induces severe thrombocytopenia caused by direct platelet demise and counteracted by increased megakaryopoiesis. In contrast, murine studies have reported important contribution of BCL-XL to survival of late erythroid cells and megakaryocytes. Using lentiviral knockdown, we show that the roles of BCL-XL for human hematopoietic cells are much more pronounced than expected from murine data and clinical trials. Efficient genetic or chemical BCL-XL inhibition resulted in significant loss of human erythroid cells beginning from very early stages of erythropoiesis, and in a reduction of megakaryocytes. Most importantly, BCL-XL deficient human hematopoietic stem cells and multipotent progenitors were reduced in numbers, and they showed a severely impaired capacity to engraft in mice during xenotransplantation. BCL-XL deficiency was fully compensated by BCL-2 overexpression, however, loss of its antagonist BIM did not result in any rescue of human erythroid or stem and progenitor cells. We thus conclude that novel and specific BCL-XL inhibitors might be efficient to treat malignancies of erythroid or megakaryocytic origin, such as polycythemia vera, acute erythroid leukemia, essential thrombocytosis or acute megakaryocytic leukemia. At the same time, it can be expected that they will have more severe hematological side effects than Navitoclax.

In vitro and in vivo evaluation of possible pro-survival activities of PGE2, EGF, TPO and FLT3L on human hematopoiesis.

Myelosuppression is a major and frequently dose-limiting side effect of anticancer therapy and is responsible for most treatment-related morbidity and mortality. In addition, repeated cycles of DNA damage and cell death of hematopoietic stem and progenitor cells, followed by compensatory proliferation and selection pressure, lead to genomic instability and pave the way for therapy-related myelodysplastic syndromes and secondary acute myeloid leukemia. Protection of hematopoietic stem and progenitor cells from chemo- and radiotherapy in patients with solid tumors would reduce both immediate complications and long-term sequelae. Epidermal growth factor (EGF) and prostaglandin E2 (PGE2) were reported to prevent chemo- or radiotherapy-induced myelosuppression in mice. We tested both molecules for potentially protective effects on human CD34+ cells in vitro and established a xenograft mouse model to analyze stress resistance and regeneration of human hematopoiesis in vivo EGF was neither able to protect human stem and progenitor cells in vitro nor to promote hematopoietic regeneration following sublethal irradiation in vivo PGE2 significantly reduced in vitro apoptotic susceptibility of human CD34+ cells to taxol and etoposide. This could, however, be ascribed to reduced proliferation rather than to a change in apoptosis signaling and BCL-2 protein regulation. Accordingly, 16,16-dimethyl-PGE2 (dmPGE2) did not accelerate regeneration of the human hematopoietic system in vivo Repeated treatment of sublethally irradiated xenograft mice with known antiapoptotic substances, such as human FLT3L and thrombopoietin (TPO), which suppress transcription of the proapoptotic BCL-2 proteins BIM and BMF, also only marginally promoted human hematopoietic regeneration in vivo.

Transient apoptosis inhibition in donor stem cells improves hematopoietic stem cell transplantation.

During hematopoietic stem cell transplantation, a substantial number of donor cells are lost because of apoptotic cell death. Transplantation-associated apoptosis is mediated mainly by the proapoptotic BCL-2 family proteins BIM and BMF, and their proapoptotic function is conserved between mouse and human stem and progenitor cells. Permanent inhibition of apoptosis in donor cells caused by the loss of these BH3-only proteins improves transplantation outcome, but recipients might be exposed to increased risk of lymphomagenesis or autoimmunity. Here, we address whether transient inhibition of apoptosis can serve as a safe but efficient alternative to improve the outcome of stem cell transplantation. We show that transient apoptosis inhibition by short-term overexpression of prosurvival BCL-XL, known to block BIM and BMF, is not only sufficient to increase the viability of hematopoietic stem and progenitor cells during engraftment but also improves transplantation outcome without signs of adverse pathologies. Hence, this strategy represents a promising and novel therapeutic approach, particularly under conditions of limited donor stem cell availability.

Nanoclusters of the resting T cell antigen receptor (TCR) localize to non-raft domains.

In the last decade an increasing number of plasma membrane (PM) proteins have been shown to be non-randomly distributed but instead forming submicron-sized oligomers called nanoclusters. Nanoclusters exist independently of the ligand-bound state of the receptors and their existence implies a high degree of lateral organisation of the PM and its proteins. The mechanisms that drive receptor nanoclustering are largely unknown. One well-defined example of a transmembrane receptor that forms nanoclusters is the T cell antigen receptor (TCR), a multisubunit protein complex whose nanoclustering influences its activity. Membrane lipids, namely cholesterol and sphingomyelin, have been shown to contribute to TCR nanoclustering. However, the identity of the membrane microdomain in which the TCR resides remains controversial. Using a GFP-labeled TCR we show here that the resting TCR localized in the disordered domain of giant PM vesicles (GPMVs) and PM spheres (PMSs) and that single and nanoclustered TCRs are found in the high-density fractions in sucrose gradients. Both findings are indicative of non-raft localization. We discuss possible mechanisms of TCR nanoclustering in T cells. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.