Research Overview

The Role of Posttranslational Modifications in Regulating Immune Checkpoint Function

Posttranslational Modifications in Genome Stability and Carcinogenesis

Posttranslational Modifications in Oncogenic Signaling

Posttranslational modifications in mitotic regulation and cancer treatment

Posttranslational modifications such as ubiquitination, methylation, ADP-ribosylation as well as phosphorylation orchestrate genome stability, cell division, hormone-initiated signal transduction, apoptosis and tumorigenesis. Posttranslational modifications act as critical molecular switches or fine-tune operators that determine the activation, deactivation or subcellular localization of functional proteins. Emerging evidence has drawn attention to the modulation of regulatory proteins in response to extrinsic/intrinsic signaling being executed simultaneously by multiple posttranslational modifications. Research interests in my laboratory seek to address how defects in the ubiquitin-proteasome system (E3 ligase/deubiquitinase), protein methyltransferase, glycosyltransferase and poly (ADP-ribose) polymerase 1 (PARP1) would result in genomic instability, abnormal cell cycle or immune checkpoint function, and aberrant signal transductions (e.g., ER, TGF-β, EGFR and Hippo) that predispose otherwise normal cells to become cancerous tumor cells. The ultimate objective is to integrate our basic research with clinical translational studies that would allow the development of new anti-cancer therapy thereby fully exploiting our knowledge of posttranslational modifications. To achieve our goals, we have developed a multidisciplinary approach that includes biochemical, cell biological, genetic, protein structural analyses as well as the use of animal models and analyses of clinical specimens.

The Role of Posttranslational Modifications in Regulating Immune Checkpoint Function

Recent studies have implied that the immune checkpoint proteins are tightly regulated by posttranslational modifications (PTMs) such as ubiquitylatin and glycosylation. PTMs are involved in regulating various aspects for immune checkpoint proteins, including their protein quality control, protein trafficking, protein stability and the immune checkpoint function on cell membrane. Our lab currently focuses on the mechanisms that regulate immune checkpoint function by PTMs and searches for new strategy for cancer therapy specifically from the view of the regulation of immune checkpoint protein by PTMs.

Despite the widespread use of immunotherapy, the poor clinical response by immune-cold tumors is a current challenge. Triple negative breast cancer (TNBC) is the most aggressive mammary carcinoma subtype. Although a PD-L1 inhibitor has been FDA approved for the treatment of metastatic TNBC, the majority of TNBC patients show limited responses, particularly when their tumors are “cold” or “non-inflamed”. In contrast with “hot” tumors, these immune-cold tumors are characterized by a scarcity of T lymphocyte infiltration, commensurate with their failure to elicit anticancer immunity. Our endeavor to identify suitable targets for improving anticancer immune responses against TNBC therapy has drawn our attention to B7-H4.

Using combinatorial approaches, we unravel a novel regulatory mechanism by which B7-H4 protein turnover is governed by asparagine (N)-linked glycosylation. N-linked glycosylation is a co- and post-translational modification important for protein stability, folding, trafficking, and physiological function. Glycosylation of B7-H4 catalyzed by specific glycosyltransferases (STT3A and UGGG1) antagonizes the B7-H4 ubiquitination by E3 ligase autocrine motility factor receptor (AMFR), thereby preventing the degradation of B7-H4 and hence stabilizing the protein. Abundant B7-H4 can inhibit the phosphorylation of eIF2α, CALR exposure, and cancer cell immunogenicity. We designed a strategy for inhibiting the oligosaccharyltransferase (OST) complex that stabilizes B7-H4, thus reducing its expression and enhancing the immunogenicity TNBC cells in the context of immunogenic cell death-eliciting chemotherapy and PD-L1-targeting immunotherapy. We are currently under the process to develop new small molecule inhibitor that could specifically target the glycosylated B7-H4 for cancer treatment. This project is funded by NIH/NCI R01 grant (CA258857 and CA258765).

Posttranslational Modifications in Genome Stability and Carcinogenesis

Kruppel-like factor 4 (KLF4) is an important regulator of cell-fate decision, including DNA damage response, inflammation, apoptosis, and stem cell renewal. Its critical impact in breast cancer formation was recently uncovered by the TCGA (The Cancer Genome Atlas) study as well as human breast cancer specimen tissue array. Surprisingly, recent studies have sketched an ambivalent nature for KLF4 in tumorigenesis as either a tissue specific tumor suppressor (in colorectal cancer) or oncogene (in breast cancer), although the underlying mechanism as to how it switches functions remains unclear. In addition, how KLF4 is regulated in response to various environmental factors such as DNA damage signal remains largely unknown. To explore the mystery, we have purified KLF4 protein complexes followed by identification of its physiological binding partners using mass spectrometry. This effort has led to the identification of several KLF4 physiological interacts including VHL (a ubiquitin ligase), PRMT5 (an arginine protein Methyltransferase) and PARP1 (poly (ADP-ribose) polymerase 1). Results from our initial characterization suggest that precise KLF4 protein levels were determined by VHL/VBC and PRMT5. While KLF4 is targeted by VHL/VBC for ubiquitylation and degradation, PRMT5-mediated methylation antagonizes KLF4 ubiquitylation thereby stabilizing KLF4. Given that KLF4 is a pivotal cellular-fate factor after exposure to DNA damage, we are now addressing how the crosstalk between ubiquitylation and methylation dictates DNA damage response by promotion of p21 and inhibition of Bax. In addition, using a protein structural and computational modeling analysis, we are elucidating the mechanisms of how PRMT5 catalyzes KLF4 methylation and how KLF4 methylation counteracts KLF4 ubiquitylation for stabilization. The ultimate goal is to develop small molecule inhibitors that could intercept PRMT5-mediated KLF4 methylation, which could be valuable for anti-breast cancer chemotherapy. This project is funded by NIH/NCI R01 grant (CA250110).

Synergism of the molecular axes PARP1-KLF4 and USP11-BRCA1 in breast cancer treatment

Our recent identification of interaction between KLF4 and PARP1 based on mass spectrometry leads to the characterization of PARP1-mediated KLF4 ADP-ribosylation in the recruitment of KLF4 to chromatin that is a critical step in ensuring KLF4-governed transcriptional function. Given the vital role of KLF4 in determining the cell-fate in response to DNA damage through regulating p21 and Bax, demonstration of KLF4 PARylation by PARP1 has revealed a new paradigm as to how cancer cells are sensitized to radiation or chemotherapeutic agents.

We are now elucidating the mechanism by which KLF4 is modified by PARylation and how ADP-ribosylation of KLF4 leads to recruitment of KLF4 onto chromatin. In addition, we recently observe that inhibition of BRCA1 deubiquitination, a mechanism that stabilizes BRCA1, results in enhanced efficacy for PARP inhibitor in killing breast cancer cells. We thus propose a synthetic lethality based strategy to treat breast cancer cells with either positive or negative BRCA1 background using combination of PARP1 and USP11 inhibitors in patient-driven breast cancer xenograft mouse model. This project is funded by NIH/NCI R01 grant (CA202963)

Proteolytic regulation of Rad17 in orchestrating checkpoint function and tumorigenesis

Genotoxic stress, such as environmental radiation and chemical mutagens, results in genomic instability leading to cancer. Both DNA-damage response and DNA repair are tightly regulated by posttranslational modifications. The pivotal role of posttranslational modification has been demonstrated in the recognition of DNA damage lesion sites, activation of DNA damage checkpoint response, recruitment of DNA repair elements and termination of DNA damage checkpoint following metabolic recovery from genotoxic stress. To systematically search for proteins that are ubiquitylated and degraded in response to genotoxic stress and to further examine their impact on genomic integrity and carcinogenesis, we have performed a high-throughput screening. One interesting candidate that we identified was Rad17, a checkpoint protein. We are currently investigating the mechanism by which Rad17 is ubiquitylated and degraded in response to DNA damage signal. By purifying protein complex followed by mass spectrometry, we recently identified functional interaction between Rad17 and Cdh1/APC (E3 ligase) as well as between Rad17 and USP20 (deubiquitinase). We are now determining how failure in proteolytic regulation of Rad17 by Cdh1/APC and USP20 would affect genomic integrity and tumorigenesis by various tumor mouse models. In collaboration with clinical group, we are developing a new combinatorial therapy for melanoma/skin cancer in synergistically targeting BRAF and ATR-Cdh1/APC-Rad17-Chk1 checkpoint pathway, using various combinations of BRAF inhibitor, ATR inhibitor, Chk1 inhibitor as well as Cdh1/Cdc20 inhibitor in an animal model.

Posttranslational Modifications in Oncogenic Signaling

Crosstalk between estrogen receptor and TGF-b signaling in breast tumor initiation, progression and invasion

Defective regulation of estrogen receptor (ER) and transforming growth factor beta (TGF-β) signaling pathways can predispose breast cells towards carcinogenesis. Our recent findings have revealed a previously unknown mechanism that centers on the interplay between KLF4, an oncogenic transcriptional factor, and VHL/VBC (E3 ligase) as well as between VHL and b-TRCP/SCF (E3 ligase) in cell cycle control and proliferation. Their functional interaction is critical in orchestrating the crosstalk between ER and TGF-β signaling pathways, which in turn determines whether breast cells retain their homeostasis or are transformed to initiate oncogenic growth. Our observations have provided insight into the pathological mystery previously observed by us and others that over 70% of human mammary cancers exhibited cellular accumulation of KLF4 and decreased levels of VHL protein and that disruption in b-TRCP function in mice could results in tumor initiation and progression. We are currently studying the molecular basis of the interplay between KLF4 and VHL/VBC as well as VHL and b-TRCP/SCF in their regulation of ER and TGF-β signal transduction and determining how impaired KLF4 proteolytic regulation due to dysregulated VHL/VBC or b-TRCP/SCF.

Killing breast tumor-initiating cell by synergizing molecular axes of PARP-KLF4 and EGFR-KLF4

PARP1, ionizing irradiation and EGF receptor signaling profoundly promote renewal of tumor-initiating cells. Synergistic blockade of PARP1-KLF4 and EGFR-KLF4 cascade could efficiently kill breast cancer cells. One of the most recent striking findings in stem cell and tumor-initiating cell field is that PARP1 could elevate iPS induction-rate by 100 folds, while ionizing irradiation significantly enhances subpopulation of breast tumor-initiating cell resulting in increases in cancer heterogeneity. We and others have observed that KLF4 is an important factor that facilitates the effect of PARP1 and ionizing irradiation. In addition, we also observed EGF2 is critical to alter subpopulation of breast tumor-initiating cell. Accordingly, we are now addressing the mechanism of how PARP1, ionizing irradiation and EGFR synergistically orchestrate KLF4 resulting in increased breast tumor-initiating cell population, and we are therefore proposing a synthetic lethality based strategy to treat breast tumor-initiating cell using combination of PARP1 and EGF2 inhibitors in human breast cancer xenograft mouse model.

Posttranslational modifications in mitotic regulation and cancer treatment

Control of G2/M transition and mitotic progression during the cell cycle is crucial for DNA damage checkpoint response and maintenance of chromosomal stability. Defects in either G2/M checkpoint or maintenance of chromosomal integrity often results in malignant cellular transformation. Development of cancer therapies through sensitizing G2/M transition or inducing mitotic catastrophe has became an attractive strategy for anti-cancer therapies. We recently identified two new players (ARID1A and USPx) that govern G2/M transition and chromosomal stability. While current deep sequencing analyses have revealed a clinical connection between genetic mutations on ARID1A with ovarian and breast cancers, our pathological studies have demonstrated a tight association between aberrant expression of USPx with breast cancer. We have recently found that both ARID1A and USPx are regulated by phosphorylation, ubiquitylation and sumoylation during cell cycle and identified such regulators upstream of ARID1A or USPx. We are currently studying the molecular mechanism by which ARID1A or USPx is regulated during transition from G2 to mitosis as well as during chromatid segregation and cytokinesis and how impaired regulation of ARID1A or USPx would impact breast carcinogenesis by using a breast cancer animal model. In addition, we are now developing chemical modulators of ARID1A or USPx that could sensitize cells to Taxol and other anti-mitotic drugs as part of a breast cancer treatment.

Systematic search for deubiquitinase that promotes breast tumor initiation, progression and metastasis

Emerging role of deubiuqitinase family members in tumor initiation, progression and invasion attracts critical attention in cancer field. To systematically search for deubiquitinase whose misexpression predisposes mammary gland epithelial cells to become cancerous or enhances breast tumor invasion, we have conducted a high throughput screening of approximately 100 deubiquitinase library. Our efforts led to the identification of USP11 as a potent oncogene with its elevated expression directly triggering malignant transformation of mammary gland epithelial cells. We further observe that USP11-driven mammary oncogenesis is due to overriding XIAP that results in antagonizing apoptosis. We are now addressing the mechanism by which XIAP is regulated by coordination between E3 ligase (XIAP acts as self-ubiquitin protein ligase) and deubiquitinase (USP11). In addition, we are determining how failure in the proteolytic regulation of XIAP by USP11 would affect cellular apoptotic feature that in turn triggers tumorigenesis by utilizing a breast cancer transgenic mouse model.

We have demonstrated the oncogenic role for KLF4 in breast carcinogenesis through regulating estrogen receptor signaling and genome stability. We have further elucidated the mechanism by which KLF4 protein stability is regulated by both VHL-mediated ubiquitylation and PRMT5- facilitated protein methylation. While VHL-VBC E3 ligase targets KLF4 for degradation, PRMT5 stabilizes KLF4 through antagonizing KLF4 ubiquitylation. Unexpected accumulation of KLF4 and PRMT5 protein are correlated with breast cancer prognosis. We thus developed small molecule inhibitor that blocks KLF4 protein methylation. In collaboration with GlaxoSmithKline Pharmaceutical Company and Northwestern Memorial Hospital, we recently opened a clinical trial that determine the synergistic effect of PRMT5 inhibitor in combing with PARP inhibitor in triple negative breast cancer treatment.

PROTACs (PROteolysis TArgeting Chimeras) has recently proved its extraordinary therapeutic potential through exceptional success on degrading estrogen and androgen receptors in cancer therapeutic. We recently utilize the PROTAC platform to develop degraders that destruct some difficult targets inhibited by the traditional inhibitor. For instance, we developed a novel chimera molecule to circumvent the challenges met by current Cdc20 small molecule inhibitors. We used Apcin-A as the warhead to target Cdc20. Regarding E3 ligase recruited for Cdc20 ubiquitylation, we have considered VHL/VBC and CRBN (Celebron), both of which have reliable binding moiety and have been applied in the design of several PROTAC molecules. To search for an optimal chemical linker maximizing the chance to form stable Cdc20-PROTAC-VHL/VBC ternary complex, we have designed and tested a series of linkers with different lengths, such as PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, and PEG9. We identify a proteolysis targeting chimera, called CP5V, which comprises the Cdc20 ligand and VHL binding moiety bridged by linker PEG5 that induces Cdc20 for degradation. We now validate the impact of CP5V using various breast cancer animal models.

Anti-Cancer Drug Development

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