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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2020 Annual Report of the Division of Intramural Research

Regulation of Mammalian Cell Proliferation and Differentiation

Melvin DePamphilis
  • Melvin L. DePamphilis, PhD, Head, Section on Eukaryotic DNA Regulation
  • Alex Vassilev, PhD, Staff Scientist
  • Xiaohong Zhang, BA, Technical Assistant
  • Arup Chakraborty, PhD, Research Fellow
  • Sushil Jaiswal, PhD, Postdoctoral Fellow
  • Ajit Roy, PhD, Postdoctoral Fellow
  • Constandina O'Connell, BS, Postbaccalaureate Fellow
  • John Oh, BS, Postbaccalaureate Fellow
  • Jack Ahrens, Summer Student

Nothing is more fundamental to living organisms than the ability to reproduce. Each time a human cell divides, it must duplicate its genome, a problem of biblical proportions. A single fertilized human egg contains 2.1 meters of DNA. An adult of about 75 kg (165 lb) consists of about 29 trillion cells containing a total of about 60 trillion meters of DNA, a distance equal to 400 times that of Earth to sun. Not only must the genome be duplicated trillions of times during human development, but it must be duplicated once and only once each time a cell divides (termed mitotic cell cycles). If we interfere with this process by artificially inducing cells to re-replicate their nuclear genome before cell division, the result is DNA damage, mitotic catastrophe, and programmed cell death (apoptosis). On rare occasions, specialized cells can duplicate their genome several times without undergoing cell division (termed endocycles), but when this occurs, it generally results in terminally differentiated polyploid cells, which are viable but no longer proliferate. However, as we age, the ability to regulate genome duplication diminishes, resulting in genome instability, which allows genetic alterations that can result in promiscuous cell division, better known as cancer.

Our research program focuses on three questions: the nature of the mechanisms that restrict genome duplication to once per cell division; how these mechanisms are circumvented to allow developmentally programmed induction of polyploidy in terminally differentiated cells; and how we can manipulate such mechanisms to destroy cancer cells selectively.

DHS (4,4′-dihydroxy-trans-stilbene) suppresses DNA replication and tumor growth by inhibiting RRM2 (ribonucleotide reductase regulatory subunit M2) [Reference 1].

The DNA replication machinery is responsible for accurate and efficient duplication of the chromosome. Given that inhibition of DNA replication can lead to replication fork stalling, resulting in DNA damage and apoptotic death, inhibitors of DNA replication are commonly used in cancer chemotherapy. Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the biosynthesis of deoxyribonucleoside triphosphates (dNTPs), which are essential for DNA replication and DNA–damage repair. Gemcitabine, a nucleotide analog that inhibits RNR, has been used to treat various cancers. However, patients often develop resistance to the drug during treatment. Thus, the development of new drugs that inhibit RNR is needed. We identified a synthetic analog of resveratrol (3,5,4′-trihydroxy-trans-stilbene), termed DHS (4,4′-dihydroxy-trans-stilbene), which acts as a potent inhibitor of DNA replication. Molecular-docking analysis identified the RRM2 (ribonucleotide reductase regulatory subunit M2) of RNR as a direct target of DHS. At the molecular level, DHS induced cyclin F–mediated down-regulation of RRM2 by the proteasome. Thus, treatment of cells with DHS reduced RNR activity and consequently decreased synthesis of dNTPs with concomitant inhibition of DNA replication, arrest of cells at S-phase, DNA damage, and finally apoptosis. In mouse models of tumor xenografts, DHS was efficacious against pancreatic, ovarian, and colorectal cancer cells. Moreover, DHS overcame both gemcitabine resistance in pancreatic cancer and cisplatin resistance in ovarian cancer. Thus, DHS is a novel anticancer agent that targets RRM2 with therapeutic potential, either alone or in combination with other agents, to arrest cancer development.

A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt several events in lysosome homeostasis [Reference 2].

High-throughput screening identified five small-molecule chemical analogs (termed the WX8-family), that disrupted three events in lysosome homeostasis: (1) lysosome fission via tubulation without preventing homotypic lysosome fusion; (2) trafficking of molecules into lysosomes without altering lysosomal acidity; and (3) heterotypic fusion between lysosomes and autophagosomes. Remarkably, the compounds did not prevent homotypic fusion between lysosomes, despite the fact that homotypic fusion required some of the same machinery essential for heterotypic fusion. The effects varied 400–fold among WX8–family members, were time- and concentration-dependent, reversible, and resulted primarily from their ability to bind specifically to the PIKFYVE phosphoinositide kinase. The ability of the WX8 family to prevent lysosomes from participating in macroautophagy/autophagy suggested that they have therapeutic potential in treating autophagy-dependent diseases. In fact, the most potent WX8 family member was 100 times more lethal to ‘autophagy-addicted’ melanoma A375 cells than the lysosomal inhibitors hydroxychloroquine and chloroquine. In contrast, cells that were insensitive to hydroxychloroquine and chloroquine were also insensitive to the WX8 family. Therefore, the WX8 family of PIKFYVE inhibitors provides a basis for developing drugs that could selectively kill autophagy-dependent cancer cells, as well as for increasing the effectiveness of established anticancer therapies through combinatorial treatments.

The Cdk2-c-Myc-miR-571 axis regulates DNA replication and genomic stability by targeting geminin [Reference 3].

DNA re-replication leads to genomic instability and has been implicated in the pathology of a variety of human cancers. Eukaryotic DNA replication is tightly controlled to ensure that it occurs only once during each cell cycle. The nuclear protein Geminin is a critical component of this control: it prevents DNA re-replication from occurring during S, G2, and early M phases by preventing MCM helicases (essential for genomic DNA replication) from forming pre-replication complexes. Geminin is targeted for degradation by the anaphase-promoting complex (APC/C) from anaphase through G1 phase. However, accumulating evidence indicates that Geminin is downregulated in late S-phase by an unknown mechanism. We used a high-throughput screen to identify microRNAs that can induce excess DNA replication and found that the microRNA miR-571 could reduce the protein level of Geminin in late S-phase independently of the APC/C. Furthermore, miR-571 regulated efficient DNA replication and S-phase cell-cycle progression. Strikingly, the transcription factor c-Myc suppressed miR-571 expression by binding directly to the miR-571 promoter. At the beginning of S-phase, the cell-cycle regulator Cdk2 (cyclin-dependent kinase 2) phosphorylated c-Myc at serine 62, promoting its association with the miR-571 promoter region. Collectively, we identified miR-571 as the first miRNA that prevents aberrant DNA replication and the Cdk2–c-Myc–miR-571 axis as a new pathway for regulating DNA replication, the cell cycle, and genomic stability in cancer cells. The significance of such finding is that they identify a novel regulatory mechanism critical for maintaining genome integrity by regulating DNA replication and cell-cycle progression.

Cell-cycle arrest and apoptosis are not dependent on p53 prior to p53–dependent embryonic stem cell differentiation [Reference 4].

Previous efforts to determine whether or not the transcription factor and tumor suppressor protein p53 is required for DNA damage–induced apoptosis in pluripotent embryonic stem cells (ESCs) produced contradictory conclusions. To resolve the issue, p53+/+ and p53–/– ESCs derived by two different methods were used to quantify time-dependent changes in nuclear DNA content: annexin-V binding; cell permeabilization; and protein expression, modification, and localization. The results revealed that the chemotherapeutic drug doxorubicin (Adriamycin [ADR]) concentrations 10 to 40 times less than commonly used in previous studies induced the DNA damage–dependent G2–checkpoint and completed apoptosis within the same time frame, regardless of the presence or absence of p53, p21, or PUMA. Elevated ADR concentrations delayed initiation of apoptosis in p53–/– ESCs, but the rates of apoptosis remained equivalent. We obtained similar results by inducing apoptosis with either staurosporine inhibition of kinase activities or WX8 disruption of lysosome homeostasis. Differentiation of ESCs by deprivation of the cytokine LIF revealed p53–dependent formation of haploid cells, increased genomic stability, and suppression of the G2–checkpoint. Minimal induction of DNA damage now resulted in p53–facilitated apoptosis, but regulation of pluripotent gene expression remained p53–independent. Primary embryonic fibroblasts underwent p53–dependent total cell-cycle arrest (a prelude to cell senescence), and p53–independent apoptosis occurred in the presence of 10–fold higher levels of ADR, consistent with previous studies. Taken together, the results reveal that the many roles of p53 in cell-cycle regulation and apoptosis are first acquired during pluripotent stem cell differentiation.

Efficacy of a small-molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non–small cell lung cancer [Reference 5]

The human positive coactivator 4 (PC4) was originally identified as a multi-functional cofactor capable of mediating transcription activation by diverse gene- and tissue-specific activators. Recent studies suggest that PC4 might also function as a novel cancer biomarker and therapeutic target for various types of cancer. siRNA knockdown studies indicated that down-regulation of PC4 expression could inhibit tumorigenicity of A549 non-small cell lung cancer tumor model in nude mice. We showed that AG-1031, a small molecule identified by high-throughput screening, can inhibit the double-stranded DNA binding activity of PC4 more effectively than PC4's single-stranded DNA binding activity. AG-1031 also specifically inhibited PC4–dependent transcriptional activation in vitro using purified transcription factors. AG-1031 inhibited proliferation of several cultured cell lines derived from non–small cell lung cancers (NSCLC) and growth of tumors that formed from A549 cell xenografts in immuno-compromised mice. Moreover, pre-injection of AG-1031 in these mice not only reduced tumor size, but also prevented tumor formation in 20% of the animals. AG-1031–treated A549 cells and tumors from AG-1031–treated animals showed a significant reduction in the levels of both PC4 and of VEGFC, a key mediator of angiogenesis in cancer. On the other hand, the weight of all tested mice remained constant during animal trials. The results demonstrated that AG-1031 is be a potential therapy for PC4-positive NSCLC.


  1. Chen CW, Li Y, Hu S, Zhou W, Meng Y, Li Z, Zhang Y, Sun J, Bo Z, DePamphilis ML, Yen Y, Han Z, Zhu W. DHS (trans-4,4'-dihydroxystilbene) suppresses DNA replication and tumor growth by inhibiting RRM2 (ribonucleotide reductase regulatory subunit M2). Oncogene 2019;38:2364-2379.
  2. Sharma G, Guardia CM, Roy A, Vassilev A, Saric A, Griner LN, Marugan J, Ferrer M, Bonifacino JS, DePamphilis ML. A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis. Autophagy 2019;15:1694-1718.
  3. Zhang Y, Li Z, Hao Q, Tan W, Sun J, Li J, Chen CW, Li Z, Meng Y, Zhou Y, Han Z, Pei H, DePamphilis ML, Zhu W. The Cdk2-c-Myc-miR-571 axis regulates DNA replication and genomic stability by targeting Geminin. Cancer Res 2019;79:4896-4910.
  4. Jaiswal SK, Oh JJ, DePamphilis ML. Cell cycle arrest and apoptosis are not dependent on p53 prior to p53-dependent embryonic stem cell differentiation. Stem Cells 2020;38:1091-1106.
  5. Zhang Y, Pavlov A, Malik S, Chen H, Kim N, Li Z, Zhang X, DePamphilis ML, Roeder RG, Ge H. Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer. PLoS One 2020;15:e0230670.


  • Juan Bonifacino, PhD, Section on Intracellular Protein Trafficking, Bethesda, MD
  • Marc Ferrer, PhD, Chemical Genomics Center, NCATS, Bethesda, MD
  • Juan Marugan, PhD, Division of Pre-Clinical Innovation, NCATS, Bethesda, MD
  • Zakir Ullah, PhD, Lahore University of Management Sciences, Lahore, Pakistan
  • Wenge Zhu, PhD, George Washington University Medical School, Washington, DC


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