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Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) has been linked to the suppression of chromosomal instability. The spindle assembly checkpoint complex is responsible for proper chromosome segregation during mitosis to prevent chromosomal instability. In this study, the novel role of PIGN as a regulator of the spindle assembly checkpoint was unveiled in leukemic patient cells and cell lines. Transient downregulation or ablation of PIGN resulted in impaired mitotic checkpoint activation due to the dysregulated expression of spindle assembly checkpoint-related proteins including MAD1, MAD2, BUBR1, and MPS1. Moreover, ectopic overexpression of PIGN restored the expression of MAD2. PIGN regulated the spindle assembly checkpoint by forming a complex with the spindle assembly checkpoint proteins MAD1, MAD2, and the mitotic kinase MPS1. Thus, PIGN could play a vital role in the spindle assembly checkpoint to suppress chromosomal instability associated with leukemic transformation and progression.

// Emmanuel K. Teye 1, 2 , Abigail Sido 1, 2 , Ping Xin 1, 2 , Niklas K. Finnberg 3 , Prashanth Gokare 3 , Yuka I. Kawasawa 4, 5, 6 , Anna C. Salzberg 4, 5, 6 , Sara Shimko 1, 2 , Michael Bayerl 7 , W. Christopher Ehmann 1, 2 , David F. Claxton 1, 2 , Witold B. Rybka 1, 2, 7 , Joseph J. Drabick 1, 2 , Hong-Gang Wang 8 , Thomas Abraham 9, 10 , Wafik S. El-Deiry 3 , Robert A. Brodsky 11 , Raymond J.Hohl 1, 2 , Jeffrey J. Pu 1, 2, 7 1 Penn State Hershey Cancer Institute, Penn State University College of Medicine, Hershey, Pennsylvania, USA 2 Department of Medicine, Penn State University College of Medicine, Hershey, Pennsylvania, USA 3 Department of Hematology/Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA 4 Institute for Personalized Medicine, Penn State University College of Medicine, Hershey, Pennsylvania, USA 5 Department of Pharmacology, Penn State University College of Medicine, Hershey, Pennsylvania, USA 6 Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey, Pennsylvania, USA 7 Department of Pathology, Penn State University College of Medicine, Hershey, Pennsylvania, USA 8 Department of Pediatrics, Penn State University College of Medicine, Hershey, Pennsylvania, USA 9 Department of Neural and Behavioral Science, Pennsylvania State University, Hershey, Pennsylvania, USA 10 Microscopy Imaging Facility, Pennsylvania State University, Hershey, Pennsylvania, USA 11 Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Correspondence to: Jeffrey J. Pu, email: jeffreypu@gmail.com Keywords: PIGN gene expression aberration, MDS, AML with myelodysplasia-related changes (AML-MRC), genomic instability, leukemogenesis Received: November 25, 2016 Accepted: January 11, 2017 Published: February 07, 2017 ABSTRACT Previous studies have linked increased frequency of glycosylphosphatidylinositol-anchor protein (GPI-AP) deficiency with genomic instability and the risk of carcinogenesis. However, the underlying mechanism is still not clear. A randomForest analysis of the gene expression array data from 55 MDS patients (GSE4619) demonstrated a significant (p = 0.0007) correlation (Pearson r =-0.4068) between GPI-anchor biosynthesis gene expression and genomic instability, in which PIGN, a gene participating in GPI-AP biosynthesis, was ranked as the third most important in predicting risk of MDS progression. Furthermore, we observed that PIGN gene expression aberrations (increased transcriptional activity but diminished to no protein production) were associated with increased frequency of GPI-AP deficiency in leukemic cells during leukemic transformation/progression. PIGN gene expression aberrations were attributed to partial intron retentions between exons 14 and 15 resulting in frameshifts and premature termination which were confirmed by examining the RNA-seq data from a group of AML patients (phs001027.v1.p1). PIGN gene expression aberration correlated with the elevation of genomic instability marker expression that was independent of the TP53 regulatory pathway. Suppression/elimination of PIGN protein expression caused a similar pattern of genomic instability that was rescued by PIGN restoration. Finally, we found that PIGN bound to the spindle assembly checkpoint protein, MAD1, and regulated its expression during the cell cycle. In conclusion, PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevents leukemic transformation/progression.

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disease caused by mutation in the PIG-A gene. When expressed, it encodes a protein that is crucial in glycophosphatidylinositol (GPI) anchor biosynthesis. The mutation of this gene causes plasma membrane GPI anchor biosynthesis deficiency, cell membrane structure alteration, signal transduction pathway blockage and vulnerability to complement complex attack. Despite these deficiencies, PNH cells have a clonal advantage and this dominant expansion is the hallmark of PNH progression. The mechanism of this growth, however, is still unknown. Recently, multiple studies demonstrated that malignant cells harboring the PIG-A mutation appear to have a higher degree of genomic instability and disease progression. This led to the proposal of using the PIG-A gene mutation frequency as the biomarker of mutagenesis to evaluate the transgenic animal mutation or mutagenicity of test compounds. The PIG-A gene mutation assay is even considered as a valuable tool for quantifying mutational events in vivo and in vitro despite the mechanism remaining unknown. This study investigated the impacts PIG-A mutation in genomic stability, DNA damage response, and DNA damage checkpoint activity, which as a measure of cellular stability as well as a possible mechanism for clonal expansion. Methods: To investigate the relationship between PIG-A gene mutation status and DNA damage checkpoint activity we looked at 45 high-risk MDS/AML patients, 3 classic PNH patients and 12 healthy controls. Additionally, the TF-1 leukemia cell line was used to evaluate PIG-A wild-type vs PIG-A CRISPR knockout vs PIG-A siRNA transient suppression. H2AX/ɣH2AX, ⍺-pChk1, ⍺-pChk2, ⍺-ubPCNA, and ⍺-pRPA proteins and RNA levels were used as a quantitative indicator of DNA damage repair activity via qTR-PCR and western blot. The comet assay and fiber combing assay were further applied to explore the DNA damage and cellular replication events. RNAseq analysis was used to explore the impact of PIG-A mutation in global gene expression. Results: The in vitro results from the leukemia cell lines with various PIG-A mutation statuses showed that the PIG-A mutation decreases ɣH2AX expression. PIG-A mutation was also associated with down-regulated expression of ⍺-pChk1, ⍺-pChk2, ⍺-ubPCNA, and ⍺-pRPA proteins. The decreased levels of those gene expressions indicate that there is increased genomic stability and less repair activity at the DNA damage response checkpoint. The results of the comet assay support this data with the PIG-A mutated cells exhibiting significantly (p In patient samples, subjects with PNH showed significantly lower levels of ɣH2AX expression when compared to both healthy controls and AML/MDS patients. The comet assay revealed that PNH patients had decreased DNA double strand breaks and fiber combing assay showed a reduced DNA repair activity when compared to normal controls and AML patients with intact PIG-A gene expression (p Additionally, RNAseq analysis reveals that the expression of 10/10 genes involved in the DNA repair mechanism were down-regulated in PIG-A mutated cells when compared to wild-type control in both cell lines and patient samples. Conclusion: The above-mentioned results showed that there were decreased expressions of both DNA damage response checkpoint genes and repair genes in both the PIG-A CRISPR knockout leukemia cell lines as well as in the PNH patients. PIG-A mutation is globally associated with reduced DNA damage response capability and increased cellular stability. Our finding explains, at least partially, why PIG-A gene mutation status could be seen as a biomarker of mutagenesis and how PNH cells dominantly expend via clonal escape. Disclosures No relevant conflicts of interest to declare.

The spindle assembly checkpoint complex (SAC) is responsible for proper chromosomal segregation during mitosis. The SAC stalls mitotic exit until proper attachment of mitotic spindles to the chromosomes and bi-orientation of the chromosomes on the spindles are achieved. Dysregulation of the SAC may result in chromosomal instability (CIN) which is known to drive leukemia progression. We previously assessed the impact of phosphatidylinositol glycan anchor biosynthesis class N (PIGN) expression aberrations on leukemia progression and showed that PIGN expression aberrations were linked with CIN and leukemia transformation in high-risk myelodysplastic syndrome (MDS) patients. An in-depth understanding of the mechanistic basis of PIGN involvement in CIN and leukemic progression would have boundless therapeutic and diagnostic implications for patients. Thus, we investigated the mechanistic link between PIGN, CIN and the SAC. PIGN downregulation via RNAi and CRISPR/Cas9 as well as ectopic overexpression studies, co-immunoprecipitation, and confocal microscopy were employed to decipher the relationship between PIGN, CIN, and SAC signaling. Additionally, we tested whether the depletion of PIGN results in aberrant cell cycle signaling and defective chromosomal segregation using flow cytometry and mitotic index assays. We initially performed cell cycle synchronization experiments using myeloid and lymphoblastoid cell lines and examined PIGN expression at different stages of the cell cycle via Western blot analyses and RT-qPCR. Our results indicated that PIGN expression was cell cycle-regulated and PIGN loss significantly impacted the expression of SAC-related proteins. CRISPR/Cas9 mediated knockout of PIGN in CD34+ mononuclear cells derived from a healthy individual resulted in the suppression of MAD1 and MAD2. A similar observation was made in HEK293 PIGN CRISPR/Cas9 knockout cells. PIGN loss in the HEK293 cells resulted in MAD1, MAD2, and MPS1 suppression but led to BUBR1 upregulation. PIGN downregulation resulted in impaired mitotic checkpoint activation and consequently impacted mitotic exit. PIGN downregulation results in defective mitotic checkpoint signaling and mitotic exit with an accumulation of missegregation errors. Interestingly, ectopic overexpression of PIGN restored the MAD1 and MAD2 expression. Co-immunoprecipitation experiments and confocal analyses in cell cycle synchronized cells respectively revealed direct interactions and co-localization between PIGN and the SAC proteins MAD1, MAD2, as well as the mitotic kinase MPS1 thus unveiling a novel spatiotemporal regulatory mechanism. PIGN physically interacts with and regulates the SAC via MAD1, MAD2, MPS1 and BUBR1 during mitotic cell cycle progression. The co-purification of PIGN with some of these mitotic checkpoint proteins showed the direct role that PIGN may play in the regulation of mitotic checkpoint signaling. Thus, PIGN as a CIN suppressor may be crucial in the regulation of mitotic integrity via the SAC as part of maintaining genome stability. Despite the ubiquity of CIN in leukemia progression, there is still limited knowledge about the mechanism(s) involved. Also, since its discovery as a CIN suppressor, the molecular mechanism by which the loss of PIGN leads to CIN has until now remained elusive. However, this study revealed a novel mechanism in which PIGN may maintain genome stability via SAC regulation. Our findings open the possibility to study PIGN as a tumor suppressor because its loss significantly altered the expression of SAC-related proteins. Ultimately, PIGN modulation could be adopted as a therapeutic approach in leukemia treatment, more specifically in the averting leukemia progression in high-risk MDS patients. Disclosures No relevant conflicts of interest to declare.

Previous studies have linked increased frequency of glycosylphosphatidylinositol-anchor protein (GPI-AP) deficiency with genomic instability and the risk of carcinogenesis. Recently, Phosphatidylinositol Glycan Anchor Biosynthesis; Class N (PIGN), a gene participating in GPI-AP synthesis, was suggested as a cancer chromosomal instability suppressor in a colon cancer model. We investigated the association of PIGN with genomic instability and leukemogenesis. A Random Forest analysis of the gene expression array data from 55 MDS patients (GSE4619) demonstrated a significant (p = 0.0007) correlation (Pearson r =-0.4068) between GPI-anchor biosynthesis gene expression and genomic instability, in which PIGN was ranked as the third most important in predicting risk of MDS progression. We observed that PIGN gene expression aberrations (increased transcriptional activity but diminished to no protein production) were associated with increased frequency of GPI-AP deficiency in leukemic cells during leukemic transformation/progression. The PIGN gene expression aberrations were attributed to partial intron retentions between exons 14 and 15 resulting in frameshifts and premature termination which were confirmed by examining the RNA-seq data from a group of AML patients (phs001027.v1.p1). PIGN gene expression aberration correlated with the elevation of genomic instability marker expression that was independent of the TP53 regulatory pathway. PIGN protein expression suppression/elimination caused a similar pattern of genomic instability that was rescued by PIGN restoration. Furthermore, PIGN bound to the spindle assembly checkpoint proteins and regulated their expression during the cell cycle. In conclusion, PIGN gene is crucial in the regulation of mitotic integrity to maintain chromosomal stability and prevents leukemic transformation/progression. Citation Format: Jeffrey J. Pu, Emmanuel K. Teye, Abigail Sido, Yuka I. Kawasawa, Ping Xin, Niklas K. Finnberg, Wafik S. El-Deiry, Sara Shimko. PIGN gene expression aberration weakens chromosomal stability via altering its interaction with the spindle assembly checkpoint protein complex during leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5713. doi:10.1158/1538-7445.AM2017-5713

Quinacrine is a bioactive acridine derivative which has been used for treatment of malaria, giardiasis, systemic lupus erythematosus, and rheumatoid arthritis. In searching for p53 pathway activating agents for cancer therapy, we found that quinacrine stabilizes p53 and induces p53-dependent and p53-independent tumor cell death. Quinacrine also induces expression of TRAIL Death Receptor 5 (DR5) and reduces expression of anti-apoptotic Mcl-1 in tumor cells. These activities predict synergies with TRAIL (tumor necrosis factor-related apoptosis inducing ligand) and chemotherapeutic agents in inducing extrinsic and intrinsic pathway mediated apoptosis. In addition, quinacrine suppresses NFkB activity in tumor cells. Clinical trials have been ongoing for treatment of solid tumors including colon cancer, renal cancer, prostate cancer, and non-small cell lung cancer with quinacrine in combination with chemotherapy or tyrosine-kinase inhibitors, however, the therapeutic potential of quinacrine in blood cancer cells has not been established. We tested quinacrine on hematopoietic malignant cells, which included cell lines of myeloid leukemia, lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and multiple myeloma. We found that quinacrine induces massive cell death in the cell lines tested, at concentrations from as less as 1 microM to 5 microM, 2-10 times lower than required to induce solid tumor cell death. Quinacrine synergizes with TRAIL in inducing cell death of TRAIL-sensitive cells and reverses resistance in TRAIL-resistant cells. Quinacrine also synergizes with chemotherapeutic agents, such as antimetabolites, alkylating agents, and tyrosine kinase inhibitors, in inducing apoptosis of hematopoietic cancer cell lines. Our work supports translational efforts to advance the use of quinacrine from bench to clinic and provides rationale for combination chemotherapeutic regimes for treatment of hematopoietic malignancies. Disclosures No relevant conflicts of interest to declare.