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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 current research program focuses on one question: how cancer cells can be selectively destroyed with little or no harm to non-malignant cells. To that end, we discovered a family of small molecules (termed the ‘WX8-family’) that selectively target the PIKFYVE phosphoinositide kinase, an enzyme that is essential for lysosome homeostasis and autophagy in cultured mammalian cells, and the PIKFYVE gene, which is essential for pre-implantation mouse development. We showed that the WX8-family of PIKFYVE inhibitors can selectively terminate autophagy-dependent cancer cells and pluripotent cancer stem cells, in culture and in tumor xenografts. The NICHD/NIH now holds a patent on the application of these molecules for cancer therapy. We described the characteristics of these molecules and their therapeutic applications in two research publications [References 2 & 5] and in one manuscript that has been accepted for publication [Roy A, Chakraborty AR, Nomanbhoy T, DePamphilis ML. PIP5K1C phosphoinositide kinase deficiency distinguishes PIKFYVE-dependent cancer cells from non-malignant cells. Autophagy 2022, accepted with revisions; journal impact factor 17], as well as in one manuscript that is in preparation [Roy A, Chakraborty AR, DePamphilis ML. PIKFYVE inhibitors induce IL24 amplified endoplasmic reticulum stress in melanoma cells and tumors].

Developmental acquisition of p53 functions

Remarkably, the p53 transcription factor, referred to as “the guardian of the genome,” is not essential for mammalian development. Moreover, efforts to identify p53–dependent developmental events have produced contradictory conclusions. Given the importance of pluripotent stem cells as models of mammalian development, and their applications in regenerative medicine and disease, resolving these conflicts is essential. We attempted to reconcile disparate data into justifiable conclusions predicated on reports that p53–dependent transcription is first detected in late mouse blastocysts, that p53 activity first becomes potentially lethal during gastrulation, and that apoptosis does not depend on p53. Furthermore, p53 does not regulate expression of genes required for pluripotency in embryonic stem cells (ESCs); it contributes to ESC genomic stability and differentiation. Depending on conditions, p53 accelerates initiation of apoptosis in ESCs in response to DNA damage, but cell-cycle arrest, as well as the rate and extent of apoptosis in ESCs, are p53–independent. In embryonic fibroblasts, p53 induces cell-cycle arrest to allow repair of DNA damage, and cell senescence to prevent proliferation of cells with extensive damage.

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. We attempted to reconcile contradictory results based on the facts that p53 cannot induce lethality in mice until gastrulation, as stated above, 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 non-canonical 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.

Combined inhibition of p38MAPK and PIKfyve synergistically disrupts autophagy to selectively target cancer cells.

In nutrient-poor conditions, autophagy buffers metabolic stress and counteracts the effects of chemotherapy and radiation on cancer cells, which depend on autophagy for survival. However, clinical trials targeting autophagy have failed to produce successful anticancer treatments using currently available inhibitors. Recent studies have shown that PIKfyve kinase inhibitors disrupt lysosome function in autophagy and can selectively kill certain cancer cells. Analysis of biochemical changes caused by PIKfyve inhibition revealed that resistant cells contain significantly elevated levels of cellular p38MAPK (p38 mitogen-activated protein kinase) protein and phosphorylation. Expression of the lysosomal protein LAMP2 (lysosome-associated membrane protein 2), carrying phospho-mimetic mutations of the p38MAPK phosphorylation sites, prevented all effects caused by PIKfyve inhibition–induced lysosome dysfunction. Thus, the activation of p38MAPK in response to PIKfyve inhibition revealed a novel compensatory role in maintaining lysosome function in autophagy. The functional cooperation between the cellular PIKfyve and p38MAPK pathways in regulating lysosome homeostasis was found to be especially important in cancer cells. Combined inhibition of PIKfyve and p38MAPK activities synergistically blocked autophagy-mediated protein degradation, prevented cathepsin maturation, and markedly reduced the viability of many cancer cell types without affecting the viability of normal cells. Furthermore, combined PIKfyve and p38MAPK inhibitors synergistically reduced tumor growth in mice bearing xenografts of human colorectal adenocarcinoma, suggesting a novel way to target cancer cells by prolonged inhibition of autophagy using lower drug concentrations. The study demonstrates that PIKfyve and p38MAPK cooperate to regulate lysosome homeostasis and that their combined inhibition synergistically blocks autophagy to reduce cancer cell viability in vitro and in vivo.

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. We confirmed the ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells 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–specific inhibitors restrict replication of many 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.

Patent

DePamphilis ML, Sharma G, Roy A, Marugan JJ, Ferrer M. Autophagy Modulators for Use in Treating Cancer: US Patent 11,471,460, October 18, 2022; for the WX8-family of PIKFYVE inhibitors described in:

1. 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;
2. Roy A, Chakraborty AR, Nomanbhoy T, DePamphilis ML. PIP5K1C Phosphoinositide kinase deficiency distinguishes PIKFYVE-dependent cancer cells from non-malignant cells. Autophagy 2022;under review;
3. References 2, 5, and 6 in this report.

Publications

1. Klionsky DJ, et al., DePamphilis ML, et al., Roy A, et al., Sharma G, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4^th edition). Autophagy 2021 17(1):1–382.
2. O'Connell CE, Vassilev A. Combined inhibition of p38MAPK and PIKfyve synergistically disrupts autophagy to selectively target cancer cells. Cancer Res 2021 81(11):2903–2917.
3. Jaiswal SK, Raj S, DePamphilis ML. Developmental acquisition of p53 functions. Genes (Basel) 2021 12(11):1675.
4. Raj S, Jaiswal SK, DePamphilis ML. Cell death and the p53 enigma during embryonic development. Stem Cells 2022 40(3):227–238.
5. 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.
6. 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.

Contact

For more information, email depamphm@mail.nih.gov or visit https://depamphilislab.nichd.nih.gov.

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