Welcome to Our Lab
Dr. Wang’s lab is part of the Division of Pediatric Hematology and Oncology in the Department of Pediatrics at Penn State College of Medicine in Hershey, Pennsylvania. The laboratory is funded by the National Institutes of Health, Four Diamonds, and Lois High Berstler and provides an excellent training environment for students and fellows who are interested in basic and translational cancer research.
Our Research
Research in this laboratory aims to better understand the fundamental mechanisms that control apoptosis (a cell self-killing mechanism) and autophagy (a cell self-eating process). The ultimate goal of Dr. Wang’s research is to translate basic science discoveries to the development of new approaches for the treatment and prevention of cancer.
Research Highlights
Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure
Ye Y, Liang X, Wang G, Bewley MC, Hamamoto K, Liu X, Flanagan JM, Wang HG, Takahashi Y, Tian F .
Commun Biol. 2024 Mar 15;7(1):334.. PMID: 38491121.
VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.
ER stress elicits non-canonical CASP8 (caspase 8) activation on autophagosomal membranes to induce apoptosis
Hattori T, Fundora KA, Hamamoto K, Opozda DM, Liang X, Liu X, Zhang J, Uzun Y, Takahashi Y, Wang HG.
Autophagy. 2023 Sep 21;1-16. PMID: 37733908.
The VPS37A gene encodes a subunit of the endosomal sorting complex required for transport (ESCRT)-I complex that is frequently lost in a wide variety of human solid cancers. We have previously demonstrated the role of VPS37A in directing the ESCRT membrane scission machinery to seal the phagophore for autophagosome completion. Here, we report that VPS37A-deficient cells exhibit an accumulation of the apoptotic initiator CASP8 (caspase 8) on the phagophore and are primed to undergo rapid apoptosis through the intracellular death-inducing signaling complex (iDISC)-mediated CASP8 activation upon exposure to endoplasmic reticulum (ER) stress. Using CRISPR-Cas9 gene editing and comparative transcriptome analysis, we identified the ATF4-mediated stress response pathway as a crucial mediator to elicit iDISC-mediated apoptosis following the inhibition of autophagosome closure. Notably, ATF4-mediated iDISC activation occurred independently of the death receptor TNFRSF10B/DR5 upregulation but required the pro-apoptotic transcriptional factor DDIT3/CHOP to enhance the mitochondrial amplification pathway for full-activation of CASP8 in VPS37A-deficient cells stimulated with ER stress inducers. Our analysis also revealed the upregulation of NFKB/NF-kB signaling as a potential mechanism responsible for restraining iDISC activation and promoting cell survival upon VPS37A depletion. These findings have important implications for the future development of new strategies to treat human cancers, especially those with VPS37A loss.
Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy
Ye Y, Tyndall ER, Bui V, Bewley MC, Wang G, Hong X, Shen Y, Flanagan JM, Wang HG*, Tian F*.
Nat Commun. 2023 Sep 7;14(1):5503. PMID: 37679347
Autophagosome formation, a crucial step in macroautophagy (autophagy), requires the covalent conjugation of LC3 proteins to the amino headgroup of phosphatidylethanolamine (PE) lipids. Atg3, an E2-like enzyme, catalyzes the transfer of LC3 from LC3-Atg3 to PEs in targeted membranes. Here we show that the catalytically important C-terminal regions of human Atg3 (hAtg3) are conformationally dynamic and directly interact with the membrane, in collaboration with its N-terminal membrane curvature-sensitive helix. The functional relevance of these interactions was confirmed by in vitro conjugation and in vivo cellular assays. Therefore, highly curved phagophoric rims not only serve as a geometric cue for hAtg3 recruitment, but also their interaction with hAtg3 promotes LC3-PE conjugation by targeting its catalytic center to the membrane surface and bringing substrates into proximity. Our studies advance the notion that autophagosome biogenesis is directly guided by the spatial interactions of Atg3 with highly curved phagophoric rims.
Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy
Liu X, Wills CA, Chen L, Zhang J, Zhao Y, Zhou M, Sundstrom JM, Schell T, Spiegelman VS, Young MM, Wang HG.
J Immunother Cancer. 2022 Apr;10(4):e004399. PMID: 35483745
In this study, we demonstrated that neuroblastoma-derived small extracellular vesicles (sEVs) significantly attenuated the efficacy of dinutuximab in vivo and modulated tumor immune cell infiltration upon dinutuximab treatment to create an immunosuppressive TME that contains more tumor-associated macrophages and fewer tumor-infiltrating NK cells. In addition, we demonstrated that neuroblastoma-derived sEVs suppress splenic NK cell maturation in vivo and dinutuximab-induced NK cell-mediated antibody-dependent cellular cytotoxicity in vitro. Importantly, tipifarnib drastically enhanced the efficacy of dinutuximab-mediated inhibition of tumor growth and prevented the immunosuppressive effects of neuroblastoma-derived sEVs in vivo. These preclinical findings uncover a novel mechanism by which neuroblastoma-derived sEVs modulate the immune system to promote resistance to dinutuximab and suggest that tipifarnib-mediated inhibition of sEV secretion may serve as a viable treatment strategy to enhance the antitumor efficacy of anti-GD2 immunotherapy in high-risk neuroblastoma patients.
Thoracic Neuroblastoma: A Novel Surgical Model for the Study of Extra-adrenal Neuroblastoma
Grant CN, Wills CA, Liu X, Spiegelman VS, Wang HG.
In Vivo. 2022 Jan-Feb;36(1):49-56. PMCID: PMC8765145
Neuroblastoma is clinically and molecularly heterogeneous, with poor outcomes despite multimodal treatment strategies. The primary tumor site is an independent predictor of survival; adrenal tumors have the worst outcomes, while posterior mediastinum tumors carry a more favorable prognosis. To elucidate the role of the primary tumor microenvironment in mediating survival outcomes, we developed a mouse model for the study of extra-adrenal neuroblastoma by injecting luciferase-tagged cells into either the subpleural space of the posterior chest or the adrenal gland. Solid tumors developed in the thoracic cavity at the same rate and efficiency as the adrenal as early as one week post-surgery. The survival rate following surgery was equivalent, though the physiological tolerance for large tumors was lower in the thoracic group. This novel mouse model of survivable extra-adrenal neuroblastoma will enable future investigations of the distinct tumor microenvironments between the adrenal gland and posterior mediastinum.
ESCRT in autophagosome closure cited as a major breakthrough in basic autophagy research
Mizushima N, White E, Rubinsztein DC.
Trend in Molecular Medicine. 2021 Sep;27(9):835-838. PMID: 34257008
Our work demonstrating the ESCRT machinery in autophagosome closure (Takahashi, Y., He, H., Tang, Z. et al. An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure. Nat Commun 2018 Jul 20;9(1):2855. PMCID: PMC6054611) was recently highlighted as one of the major breakthroughs in the field of basic macroautophagy research.
Chemotherapy-Induced Upregulation of Small Extracellular Vesicle-Associated PTX3 Accelerates Breast Cancer Metastasis
Wills CA, Liu X, Chen L, Zhao Y, Dower CM, Sundstrom J, Wang HG.
Cancer Res. 2021 Jan 15;81(2):452-463. PMCID: PMC7855036
Although neoadjuvant chemotherapy is a standard component of breast cancer treatment, recent evidence suggests that chemotherapeutic drugs can promote metastasis through poorly-defined mechanisms. Here we utilize xenograft mouse models of triple-negative breast cancer to explore the importance of chemotherapy-induced tumor-derived small extracellular vesicles (sEV) in metastasis. Doxorubicin (DXR) enhanced tumor cell sEV secretion to accelerate pulmonary metastasis by priming the pre-metastatic niche. Proteomic analysis and CRISPR/Cas9 gene editing identified the inflammatory glycoprotein PTX3 enriched in DXR-elicited sEV as a critical regulator of chemotherapy-induced metastasis. Both genetic inhibition of sEV secretion from primary tumors and pharmacologic inhibition of sEV uptake in secondary organs suppressed metastasis following chemotherapy. Taken together, this research uncovers a mechanism of chemotherapy-mediated metastasis by which drug-induced upregulation of sEV secretion and PTX3 protein cargo primes the pre-metastatic niche and suggests that inhibition of either sEV uptake in secondary organs or secretion from primary tumor cells may be promising therapeutic strategies to suppress metastasis.
Targeting the ESCRT-III component CHMP2A for noncanonical Caspase-8 activation on autophagosomal membranes
Hattori T, Takahashi Y, Chen L, Tang Z, Wills CA, Liang X, Wang HG.
Cell Death Differ. 2021 Feb;28(2):657-670. PMCID: PMC7862398
Autophagosomal membranes can serve as activation platforms for intracellular death-inducing signaling complexes (iDISCs) to initiate Caspase-8-dependent apoptosis. In this study, we explore the impact of ESCRT-III-dependent phagophore closure on iDISC assemblies and cell death in osteosarcoma and neuroblastoma cells. Inhibition of phagophore closure by conditional depletion of CHMP2A, an ESCRT-III component, stabilizes iDISCs on immature autophagosomal membranes and induces Caspase-8-dependent cell death. Importantly, suppression of the iDISC formation via deletion of ATG7, an E1 enzyme for ubiquitin-like autophagy-related proteins, blocks Caspase-8 activation and cell death following CHMP2A depletion. Although DR5 expression and TRAIL-induced apoptosis are enhanced in CHMP2A-depleted cells, the canonical extrinsic pathway of apoptosis is not responsible for the initiation of cell death by CHMP2A depletion. Furthermore, the loss of CHMP2A impairs neuroblastoma tumor growth associated with decreased autophagy and increased apoptosis in vivo. Together, these findings indicate that inhibition of the ESCRT-III-dependent autophagosome sealing process triggers noncanonical Caspase-8 activation and apoptosis, which may open new avenues for therapeutic targeting of autophagy in cancer.
Glucocorticoids enhance the anti-leukemic activity of FLT3 inhibitors in FLT3 mutant acute myeloid leukemia
Gebru MT, Atkinson JM, Young MM, Zhang L, Tang Z, Liu Z, Lu P, Dower CM, Chen L, Annageldiyev C, Sharma A, Imamura Kawasawa Y, Zhao Z, Miller BA, Claxton DF, Wang HG.
Blood. 2020 Aug 27;136(9):1067-1079. PMCID: PMC7453151
FLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug-resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained sub-population of drug-sensitive cells, so-called drug-tolerant persisters (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA-sequencing and drug-screening, here we report that treatment of FLT3-ITD AML cells with quizartinib, a selective FLT3 inhibitor, up-regulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids. Mechanistically, the combination of FLT3 inhibitors and glucocorticoids enhances cell death of FLT3 mutant but not wild-type cells through glucocorticoid receptor-dependent upregulation of the pro-apoptotic protein BIM and proteasomal degradation of the anti-apoptotic protein MCL-1. Moreover, the enhanced anti-leukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and glucocorticoids has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3 mutant AML.
TOM40 Targets Atg2 to Mitochondria-Associated ER Membranes for Phagophore Expansion.
Tang Z, Takahashi Y, He H, Hattori T, Chen C, Liang X, Chen H, Young MM, Wang HG.
Cell Rep. 2019 Aug 13; 28(7):1744-1757.e5. PMCID: PMC6701867
During autophagy, phagophores grow into double-membrane vesicles called autophagosomes, but the underlying mechanism remains unclear. Here, we show a critical role of Atg2A in phagophore expansion. Atg2A translocates to the phagophore at the mitochondria-associated ER membrane (MAM) through a C-terminal 45-amino acid domain that we have termed the MAM localization domain (MLD). Proteomic analysis identifies the outer mitochondrial membrane protein TOM40 as a MLD-interacting partner. The Atg2A-TOM40 interaction is responsible for MAM localization of Atg2A and requires the TOM receptor protein TOM70. In addition, Atg2A interacts with Atg9A by a region within its N terminus. Inhibition of either Atg2A-TOM40 or Atg2A-Atg9A interactions impairs phagophore expansion and accumulates Atg9A-vesicles in the vicinity of autophagic structures. Collectively, we propose a model that the TOM70-TOM40 complex recruits Atg2A to the MAM for vesicular and/or non-vesicular lipid transport into the expanding phagophore to grow the size of autophagosomes for efficient autophagic flux.
VPS37A directs ESCRT recruitment for phagophore closure
Takahashi Y, Liang X, Hattori T, Tang Z, He H, Chen H, Liu X, Abraham T, Imamura-Kawasawa Y, Buchkovich NJ, Young MM, Wang HG.
J Cell Biol. 2019 Oct 7;218(10):3336-3354. PMCID: PMC6781443
The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.
An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure.
Takahashi Y, He H, Tang Z, Hattori T, Liu Y, Young MM, Serfass JM, Chen L, Gebru M, Chen C, Wills CA, Atkinson JM, Chen H, Abraham T, Wang HG.
Nat Commun. 2018 Jul 20;9(1):2855. PMCID: PMC6054611
The mechanism of phagophore closure remains unclear due to technical limitations in distinguishing unclosed and closed autophagosomal membranes. Here, we report the HaloTag-LC3 autophagosome completion assay that specifically detects phagophores, nascent autophagosomes, and mature autophagic structures. Using this assay, we identify the endosomal sorting complexes required for transport (ESCRT)-III component CHMP2A as a critical regulator of phagophore closure. During autophagy, CHMP2A translocates to the phagophore and regulates the separation of the inner and outer autophagosomal membranes to form double-membrane autophagosomes. Consistently, inhibition of the AAA-ATPase VPS4 activity impairs autophagosome completion. The ESCRT-mediated membrane abscission appears to be a critical step in forming functional autolysosomes by preventing mislocalization of lysosome-associated membrane glycoprotein 1 to the inner autophagosomal membrane. Collectively, our work reveals a function for the ESCRT machinery in the final step of autophagosome formation and provides a useful tool for quantitative analysis of autophagosome biogenesis and maturation.
Website created and maintained by Dr. Megan Young (myoung3@pennstatehealth.psu.edu).