Na Yuan

Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia is the most common type of pediatric leukemia. Despite improved remission rates, current treatment regimens for pediatric B-cell acute lymphoblastic leukemia are often associated with adverse effects and central nervous system relapse, necessitating more effective and safer agents. Bafilomycin A1 is an inhibitor of vacuolar H+-ATPase that is frequently used at high concentration to block late-phase autophagy. Here, we show that bafilomycin A1 at a low concentration (1 nM) effectively and specifically inhibited and killed pediatric B-cell acute lymphoblastic leukemia cells. It targeted both early and late stages of the autophagy pathway by activating mammalian target of rapamycin signaling and by disassociating the Beclin 1-Vps34 complex, as well as by inhibiting the formation of autolysosomes, all of which attenuated functional autophagy. Bafilomycin A1 also targeted mitochondria and induced caspase-independent apoptosis by inducing the translocation of apoptosis-inducing factor from mitochondria to the nucleus. Moreover, bafilomycin A1 induced the binding of Beclin 1 to Bcl-2, which further inhibited autophagy and promoted apoptotic cell death. In primary cells from pediatric patients with B-cell acute lymphoblastic leukemia and a xenograft model, bafilomycin A1 specifically targeted leukemia cells while sparing normal cells. An in vivo mouse toxicity assay confirmed that bafilomycin A1 is safe. Our data thus suggest that bafilomycin A1 is a promising candidate drug for the treatment of pediatric B-cell acute lymphoblastic leukemia.

Loss of autophagy leads to failure in megakaryopoiesis, megakaryocyte differentiation, and thrombopoiesis in mice

During hematopoiesis, megakaryopoiesis, megakaryocyte differentiation, and thrombopoiesis are regulated at multiple stages, which involve successive lineage commitment steps and proceed with polyploidization, maturation, and organized fragmentation of the cytoplasm, leading to the release of platelets in circulation. However, the cellular mechanisms by which megakaryocytes derive from their progenitors and differentiate into platelets have not fully been understood. Using an Atg7 hematopoietic conditional knockout mouse model, we found that loss of autophagy, a metabolic process essential in homeostasis and cellular remodeling, caused mitochondrial and cell cycle dysfunction, impeding megakaryopoiesis and megakaryocyte differentiation, as well as thrombopoiesis and subsequently produced abnormal platelets, larger in size and fewer in number, ultimately leading to severely impaired platelet production and failed hemostasis.

Autophagy confers DNA damage repair pathways to protect the hematopoietic system from nuclear radiation injury

Autophagy is essentially a metabolic process, but its in vivo role in nuclear radioprotection remains unexplored. We observed that ex vivo autophagy activation reversed the proliferation inhibition, apoptosis, and DNA damage in irradiated hematopoietic cells. In vivo autophagy activation improved bone marrow cellularity following nuclear radiation exposure. In contrast, defective autophagy in the hematopoietic conditional mouse model worsened the hematopoietic injury, reactive oxygen species (ROS) accumulation and DNA damage caused by nuclear radiation exposure. Strikingly, in vivo defective autophagy caused an absence or reduction in regulatory proteins critical to both homologous recombination (HR) and non-homologous end joining (NHEJ) DNA damage repair pathways, as well as a failure to induce these proteins in response to nuclear radiation. In contrast, in vivo autophagy activation increased most of these proteins in hematopoietic cells. DNA damage assays confirmed the role of in vivo autophagy in the resolution of double-stranded DNA breaks in total bone marrow cells as well as bone marrow stem and progenitor cells upon whole body irradiation. Hence, autophagy protects the hematopoietic system against nuclear radiation injury by conferring and intensifying the HR and NHEJ DNA damage repair pathways and by removing ROS and inhibiting apoptosis.

Autophagy regulates the cell cycle of murine HSPCs in a nutrient-dependent manner

Autophagy is implicated in hematopoiesis, but its role in the regulation of the hematopoietic stem and progenitor cell (HSPC) cycle remains obscure. Here, we show that autophagy is essential to maintain and regulate the cell cycle of HSPCs in a nutrient-dependent manner. The loss of autophagy via conditional deletion of its essential gene atg7 ablated the cell cycle of HSPCs. Under physiologic or nutrient-rich conditions, the in vivo activation of autophagy promoted the cell cycle entry of hematopoietic stem cells and upregulated cyclin D3 expression in HSPCs. In contrast, under poor nutrient conditions, the ex vivo inhibition of early, but not late, autophagy signaling events enhanced the G1/S transition of HSPCs. Cyclin D3 was downregulated in HSPCs in response to nutrient stress. A knockdown of cyclin D3 blocked the G1/S transition in HSPCs. Surprisingly, the ex vivo inhibition of starvation-induced early, but not late, autophagy signaling inhibited the ubiquitin-mediated degradation of cyclin D3 in HSPCs. Furthermore, a conditional autophagy defect also had this effect, leading to an elevated cyclin D3 level in these cells. This suggested a dependency of the proteasomal degradation of cyclin D3 on early, but not late, events in autophagy signaling under nutrient stress in HSPCs. Our results thus indicate a dual role of autophagy in the nutrient-dependent modulation of the cell cycle entry of hematopoietic stem cells and G1/S transition of HSPCs via the regulation of cyclin D3 to maintain a proper cell cycle in HSPCs and normal hematopoiesis during adult life.

Hierarchal autophagic divergence of hematopoietic system.

Background: Autophagy is required in hematopoiesis and protects against leukemogenesis. Results: When ATG7-dependent canonical autophagy is impaired, ATG7-independent alternative autophagy engages in myeloid cells but not in hematopoietic stem cells. Conclusion: The integrity of hematopoietic stem cells is jeopardized by a lack of alternative autophagy. Significance: Learning autophagy organization in hematopoietic system is crucial for understanding hematopoietic stem cell transformation. Autophagy is integral to hematopoiesis and protects against leukemogenesis. However, the fundamentals of the required molecular machinery have yet to be fully explored. Using conditional mouse models to create strategic defects in the hematopoietic hierarchy, we have shown that recovery capacities in stem cells and somatic cells differ if autophagy is impaired or flawed. An in vivo Atg7 deletion in hematopoietic stem cells completely ablates the autophagic response, leading to irreversible and ultimately lethal hematopoiesis. However, while no adverse phenotype is manifested in vivo by Atg7-deficient myeloid cells, they maintain active autophagy that is sensitive to brefeldin A, an inhibitor targeting Golgi-derived membranes destined for autophagosome formation in alternative autophagy. Removing Rab9, a key regulatory protein, in alternative autophagy, disables autophagy altogether in Atg7-deficient macrophages. Functional analysis indicates that ATG7-dependent canonical autophagy is physiologically active in both hematopoietic stem cells and in terminally differentiated hematopoietic cells; however, only terminally differentiated cells such as macrophages are rescued by alternative autophagy if canonical autophagy is ineffective. Thus, it appears that hematopoietic stem cells rely solely on ATG7-dependent canonical autophagy, whereas terminally differentiated or somatic cells are capable of alternative autophagy in the event that ATG7-mediated autophagy is dysfunctional. These findings offer new insight into the transformational trajectory of hematopoietic stem cells, which in our view renders the autophagic machinery in stem cells more vulnerable to disruption.

Role of autophagy in acute myeloid leukemia therapy.

Despite its dual role in determining cell fate in a wide array of solid cancer cell lines, autophagy has been robustly shown to suppress or kill acute myeloid leukemia cells via degradation of the oncogenic fusion protein that drives leukemogenesis. However, autophagy also induces the demise of acute leukemia cells that do not express the known fusion protein, though the molecular mechanism remains elusive. Nevertheless, since it can induce cooperation with apoptosis and differentiation in response to autophagic signals, autophagy can be manipulated for a better therapy on acute myeloid leukemia.

Erythroleukemia cells acquire an alternative mitophagy capability

Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7−/− leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor brefeldin A or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy

Beclin 1 is a well-established core mammalian autophagy protein that is embryonically indispensable and has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Here, we show that Beclin 1 is a prenatal primary cytoplasmic protein but rapidly relocated into the nucleus during postnatal development in mice. Surprisingly, deletion of beclin1 in in vitro human cells did not block an autophagy response, but attenuated the expression of several DNA double-strand break (DSB) repair proteins and formation of repair complexes, and reduced an ability to repair DNA in the cells exposed to ionizing radiation (IR). Overexpressing Beclin 1 improved the repair of IR-induced DSB, but did not restore an autophagy response in cells lacking autophagy gene Atg7, suggesting that Beclin 1 may regulate DSB repair independent of autophagy in the cells exposed to IR. Indeed, we found that Beclin 1 could directly interact with DNA topoisomerase IIβ and was recruited to the DSB sites by the interaction. These findings reveal a novel function of Beclin 1 in regulation of DNA damage repair independent of its role in autophagy particularly when the cells are under radiation insult.

Autophagy Promotes the Repair of Radiation-Induced DNA Damage in Bone Marrow Hematopoietic Cells via Enhanced STAT3 Signaling

Autophagy protects hematopoietic cells from radiation damage in part by promoting DNA damage repair. However, the molecular mechanisms by which autophagy regulates DNA damage repair remain largely elusive. Here, we report that this radioprotective effect of autophagy depends on STAT3 signaling in murine bone marrow mononuclear cells (BM-MNCs). Specifically, we found that STAT3 activation and nuclear translocation in BM-MNCs were increased by activation of autophagy with an mTOR inhibitor and decreased by knockout of the autophagy gene Atg7. The autophagic regulation of STAT3 activation is likely mediated by induction of KAP1 degradation, because we showed that KAP1 directly interacted with STAT3 in the cytoplasm and knockdown of KAP1 increased the phosphorylation and nuclear translocation of STAT3. Subsequently, activated STAT3 transcriptionally upregulated the expression of BRCA1, which increased the ability of BM-MNCs to repair radiation-induced DNA damage. This novel finding that activation of autophagy can promote DNA damage repair in BM-MNCs via the ATG-KAP1-STAT3-BRCA1 pathway suggests that autophagy plays an important role in maintaining genomic integrity of BM-MNCs and its activation may confer protection of BM-MNCs against radiation-induced genotoxic stress.

Autophagy maintains ubiquitination-proteasomal degradation of Sirt3 to limit oxidative stress in K562 leukemia cells.

Sirtuin protein family member 3 (Sirt3) has been suggested as a positive regulator in alleviating oxidative stress by acting on the mitochondrial antioxidant machinery in solid tumors; however, its role and regulation in hematological malignancies has been poorly understood. Here, we show that contrary to what has been reported in solid tumors, in K562 leukemia cells elevated Sirt3 was associated with mitochondrial stress, and depletion of Sirt3 decreased reactive oxygen species (ROS) generation and lipid oxidation, but increased the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), suggesting an opposite role of Sirt3 in regulating oxidative stress in the leukemia cells. Notably, loss of autophagy by deletion of autophagy essential gene or by pharmacological inhibition on autophagic degradation caused a significant accumulation of Sirt3. However, induced activation of autophagy did not cause autophagic degradation of Sirt3. Furthermore, inhibiting proteasome activity accumulated Sirt3 in autophagy-intact but not autophagy-defective cells, and disrupting functional autophagy either genetically or pharmacologically caused significantly less ubiquitination of Sirt3. Therefore, our data suggest that basal but not enhanced autophagy activity maintains ubiquitination-proteasomal degradation of Sirt3 to limit lipid oxidative stress, representing an adaptive mechanism by which autophagy, in collaboration with the ubiquitination-proteasomal system, controls oxidative stress by controlling the levels of certain proteins in K562 leukemia cells.