Regulation of Mammalian Cell Proliferation and Differentiation
- Melvin L. DePamphilis, PhD, Head, Section on Eukaryotic DNA Regulation
- Arup Chakraborty, PhD, Research Fellow
- Ajit Roy, PhD, Postdoctoral Fellow
Our research initially focused on mechanisms that restrict nuclear DNA replication during cell division to one complete copy of the genome during each of the trillions of cell divisions required for fertilized mammalian eggs to develop into adults. These studies led to discoveries in four areas. First, they elucidated the mechanisms by which trophoblast stem cells and megakaryoblasts are developmentally programmed to differentiate into nonproliferating polyploid cells via endoreplication, a process in which mitosis is bypassed and a second S-phase ensues. Second, they identified genes that are essential in non-malignant cells to prevent unscheduled endoreplication from promoting cancer, as well as genes essential to prevent normal cells from re‑replicating their DNA more than once during a single S-phase. Third, these studies led to the discovery that induction of DNA re-replication during the same cell cycle can selectively kill cancer cells with little or no harm to normal cells. Our efforts to identify small molecules that could selectively induce DNA re-replication in cancer cells resulted in the discovery of a family of PIKFYVE phosphoinositide kinase inhibitors that can selectively kill autophagy-dependent cancer cells. Current research focuses on distinguishing sensitive cells from insensitive cells, the mechanism by which PIKFYVE inhibitors kill cancer cells, and the therapeutic potential of PIKFYVE inhibitors.
PIP5K1C phosphoinositide kinase deficiency distinguishes PIKFYVE–dependent cancer cells from non-malignant cells.
Although PIKFYVE phosphoinositide kinase inhibitors can selectively eliminate PIKFYVE–dependent human cancer cells in vitro and in vivo, the basis for this selectivity has remained elusive. We showed that the sensitivity of cells to the PIKFYVE inhibitor WX8 is not linked to PIKFYVE expression, macroautophagic/autophagic flux, the BRAFV600E mutation, or ambiguous inhibitor specificity. PIKFYVE dependence results from a deficiency in the PIP5K1C phosphoinositide kinase, an enzyme required for the conversion of phosphatidylinositol-4-phosphate (PtdIns4P) into phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2/PIP2), a phosphoinositide associated with lysosome homeostasis, endosome trafficking, and autophagy. PtdIns(4,5)P2 is produced via two independent pathways: one requires PIP5K1C; the other requires PIKFYVE and PIP4K2C to convert PtdIns3P into PtdIns(4,5)P2. In PIKFYVE–dependent cells, low concentrations of WX8 specifically inhibit PIKFYVE in situ, thereby increasing the level of its substrate PtdIns3P, while suppressing PtdIns(4,5)P2 synthesis and inhibiting lysosome function and cell proliferation. At higher concentrations, WX8 inhibits both PIKFYVE and PIP4K2C in situ, which amplifies these effects to further disrupt autophagy and induce cell death. WX8 did not alter PtdIns4P levels. Consequently, inhibition of PIP5K1C in WX8–resistant cells transformed them into sensitive cells, and overexpression of PIP5K1C in WX8–sensitive cells increased their resistance to WX8. The discovery suggests that PIKFYVE–dependent cancers could be identified clinically by low levels of PIP5K1C and treated with PIKFYVE inhibitors.
PIKfyve–specific inhibitors restrict replication of multiple coronaviruses in vitro but not in a murine model of COVID-19.
The ongoing COVID-19 pandemic has claimed more than 6 million lives and continues to test the world economy and healthcare systems. To combat this pandemic, the biological research community has shifted efforts to the development of medical countermeasures, including vaccines and therapeutics. However, to date, the only small molecules approved for the treatment of COVID-19 in the United States are the nucleoside analogue Remdesivir and the protease inhibitor Paxlovid, although several compounds have received Emergency Use Authorization and many more are currently being tested in human efficacy trials. One such compound, Apilimod, is being considered as a COVID-19 therapeutic in a Phase II efficacy trial. However, at the time of writing, there were no published efficacy data in human trials or animal COVID-19 models. We showed that, while Apilimod and other PIKfyve inhibitors have potent antiviral activity in various cell lines against multiple human coronaviruses, these compounds worsen disease in a COVID-19 murine model, when given prophylactically or therapeutically.
Selective elimination of pluripotent stem cells by PIKfyve–specific inhibitors
Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. We showed that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve–specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. The results provide a proof of principle that PIKfyve–specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.
PIKFYVE inhibitors trigger interleukin-24–dependent termination of autophagy-dependent melanoma cells and tumors.
PIKFYVE phosphoinositide kinase–specific inhibitors disrupt lysosome homeostasis, thereby selectively terminating autophagy-dependent cancer cells in vivo as well as in vitro without harming the viability of non-malignant cells. To elucidate the mechanism by which PIKFYVE inhibition induces cell death, PIKFYVE–dependent melanoma cells were compared with PIKFYVE–independent foreskin fibroblasts. RNA sequence profiling suggested that PIKFYVE inhibitors upregulated an endoplasmic reticulum (ER) stress response involving interleukin‑24 (IL24/mda‑7) selectively in melanoma cells. Subsequent biochemical and genetic analyses confirmed these results and extended them to tumor xenografts in which tumor formation and expansion were inhibited. IL24 gene expression was induced by the DDIT3/CHOP transcription factor, a component of the PERK (protein kinase R-like ER kinase)–dependent ER–stress response. Ectopic expression of the IL24 gene induced cell death in melanoma cells, but not in foreskin fibroblasts, whereas ablation of the IL24 gene in melanoma cells prevented death. Thus, unlike thapsigargin and tunicamycin, which induce ER stress indiscriminately, PIKFYVE inhibitors selectively terminated PIKFYVE–dependent melanoma by inducing IL24–dependent ER stress. Moreover, induction of cell death by a PIKFYVE inhibitor together with ectopic expression of IL24 protein was cumulative, thereby confirming the therapeutic potential of PIKFYVE inhibitors in the treatment of melanoma, the deadliest form of skin cancer.
Cell death and the p53 enigma during mammalian embryonic development
Twelve forms of programmed cell death (PCD) have been described in mammalian cells, but which of them occurs during embryonic development and the role played by the p53 transcription factor and tumor suppressor remains enigmatic. Although p53 is not required for mouse embryonic development, some studies conclude that PCD in pluripotent embryonic stem cells from mice (mESCs) or humans (hESCs) is p53–dependent, whereas others conclude that it is not. Given the importance of pluripotent stem cells as models of embryonic development and their applications in regenerative medicine, resolving this enigma is essential. Our review [Reference 1] reconciles contradictory results based on the facts that p53 cannot induce lethality in mice until gastrulation and that experimental conditions could account for differences in results with ESCs. Consequently, activation of the G2–checkpoint in mouse ESCs is p53–independent and generally, if not always, results in noncanonical apoptosis. Once initiated, PCD occurs at equivalent rates and to equivalent extents regardless of the presence or absence of p53. However, depending on experimental conditions, p53 can accelerate initiation of PCD in ESCs and late-stage blastocysts. In contrast, DNA damage following differentiation of ESCs in vitro or formation of embryonic fibroblasts in vivo induces p53–dependent cell cycle arrest and senescence.
Patent
Autophagy Modulators for Use in Treating Cancer: US Patent 11,471,460, October 18, 2022; for the WX8-family of PIKFYVE inhibitors described in: 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(10):1694-1718.
Publications
- Raj S, Jaiswal SK, DePamphilis ML. Cell death and the p53 enigma during embryonic development. Stem Cells 2022 40(3):227–238.
- Chakraborty AR, Vassilev A, Jaiswal SK, O’Connell CE, Ahrens JF, Mallon BS, Pera MF, DePamphilis ML. Selective elimination of pluripotent stem cells by PIKfyve specific inhibitors. Stem Cell Rep 2022 17(2):397–412
- Logue J, Chakraborty AR, Johnson R, Goyal G, Rodas M, Taylor LJ, Baracco L, McGrath ME, Haupt R, Furlong BA, Soong M, Prabhala P, Horvath V, Carlson KE, Weston S, Ingber DE, DePamphilis ML, Frieman MB. PIKfyve-specific inhibitors restrict replication of multiple coronaviruses in vitro but not in a murine model of COVID-19. Commun Biol 2022 5(1):808.
- Roy A, Chakraborty AR, Nomanbhoy T, DePamphilis ML. PIP5K1C phosphoinositide kinase deficiency distinguishes PIKFYVE-dependent cancer cells from non-malignant cells. Autophagy 2023 19(9):2464–2484.
- Roy A, Chakraborty AR, DePamphilis ML. PIKFYVE inhibitors trigger interleukin-24 dependent termination of autophagy-dependent melanoma cells and tumors. Mol Oncol 2023; in press.
Contact
For more information, email depamphm@mail.nih.gov.
