MDM2 and DNMT1 inhibitors induce neuroblastoma cell death through p53-dependent and independent pathways.

Introduction: Neuroblastoma, a prevalent and aggressive pediatric cancer, poses significant treatment challenges due to its aggressive nature, rapid proliferation, and tumor heterogeneity, particularly in high-risk cases. While genetic mutations and chromosomal abnormalities play a key role in the development and progression of neuroblastoma, there is growing evidence to confirm that epigenetic changes are equally important in influencing tumor behavior, treatment responses, and patient outcomes. Emerging evidence indicates that the DNMTs inhibitor (CM-272) may facilitate cell cycle arrest and induce apoptosis by modulating critical regulatory pathways. Therefore, our hypothesis is that CM-272 treatment could lead to apoptosis mediated cell death in SK-N-SH neuroblastoma cells through the upregulation of p21 transcription. Methods: To test our hypothesis, SK-N-SH cells were treated with 2 µM of CM-272 with untreated cells serving as control. Cell viability was evaluated using the Trypan Blue Dye Exclusion (TBDE) assay. Both qRT-PCR and western blot analyses were conducted to assess the expression levels of cell cycle, apoptotic, and epigenetic markers such as p53, p21, PARP, BAX, BCL-XL and DNMT1. The RT2 Profiler PCR array method was used for determining the expression profile of the cell cycle pathway genes. Results: Our study results confirmed that SK-N-SH cells treated with CM-272 undergo increased cell death. Consistent with our hypothesis, we observed an increase and decrease in BAX, BCL-XL levels, respectively, suggesting that DNMT1 inhibition might have facilitated the induction of apoptosis through a p21-driven mechanism. Conclusion: Our results highlight the potential use of CM-272 to induce apoptosis in SK-N-SH cells through p21 upregulation and an increase in BAX levels. Furthermore, our findings suggest that targeting DNMT1 may offer an alternative pathway for inducing cell death through elevation of p21 in neuroblastoma that may have p53 mutations. If validated in further studies, DNMTs-targeted therapies could represent a significant advancement in developing new treatments for aggressive cancers. Acknowledgements: This project was supported by the National Pediatric Cancer Foundation (NPCF). This research was also partially funded by the Bankhead Coley Infrastructure Development Grant from the Florida Department of Health that was awarded to A.R. through the Department of Pharmacology of the University of Miami (Coral Gables, Florida USA). The authors would like to thank Royal Dames of Cancer Research Inc. (Ft. Lauderdale, Florida) for their financial support in conducting this research. Citation Format: Arun Jagarlamudi, Nicolas Tapia Stoll, Shyam Sundar Jaganathan, Umamaheswari Natarajan, Appu Rathinavelu. Epigenetic modification induced by DNMTs inhibitor enhances apoptosis through p21 pathway in neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 445.

Background: Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, with amplification of the oncogene MYCN being a hallmark of high-risk disease and poor prognosis. Most NB tumors initially respond to chemotherapy; however, over half of these cases relapse, resulting in chemoresistant disease. In this stage of NB there is evidence of p53 inactivation, which suggests a role for p53 in chemo-sensitivity. Although highly mutated in other cancer types, p53 mutations in NB are rare. Previous data have shown that amplification or deregulation of MDM2, a negative regulator of p53, may be the more common mechanism of p53 suppression and chemoresistance in NB. MYCN and MDM2 have been shown to interact and contribute to NB growth and disease progression. In vitro treatment of NB cells with MDM2 inhibitors has shown promise in increasing the expression of p53, leading to a decrease in proliferation, and increasing apoptosis. BET (Bromodomain and Extra-Terminal domain) inhibitors have also been shown to be effective in treating NB cells in vitro, decreasing MYCN expression, and resulting in increased apoptosis and differentiation. We hypothesize that the combination treatment of a MDM2 inhibitor (CGM097) with a BET inhibitor (OTX015) will result in greater p53 activation and a subsequent synergistic increase in NB cell death. Methods: Single-agent IC50 values for both CGM097 and OTX015 were obtained for the established NB cell lines SMS-KCNR, SH-SY5Y, BE(2)-C, and CHLA-90 using Calcein AM and Cell Titer-Glo cell viability assays after 48 hours of treatment. Drug combination studies were performed to evaluate synergism between CGM097 and OTX015 in the treatment of NB cells. IncuCyte ZOOM live cell imaging was used for kinetic monitoring of apoptosis with single-agent and combination drug treatment in NB cells. Western blot analysis was used to determine protein levels of apoptosis (Caspase-3) and MYCN/p53 pathway targets (MYCN, MDM2 and p53). Results: IC50 values for the four established NB cell lines ranged from 0.384 µM-13.5 µM and 7.25 µM-42.79 µM for CGM097 and OTX015, respectively. The combination treatment exhibited synergistic effects in wild-type p53 NB cells, with supporting data obtained from IncuCyte imaging tracking real-time cell death. IncuCyte showed induction of caspase-mediated apoptosis confirmed by Western blot analysis of Cleaved Caspase 3. Pathway analysis shows the importance of wild-type p53 for treatment effect. Conclusion: This study indicates that the combination of CGM097 and OTX015 synergistically decreases viability in NB cells with wild-type p53 expression. Further in vitro and in vivo work will be necessary to elucidate the mechanisms behind this interaction and in vivo efficacy. Citation Format: Tyler P. Maser, Joseph W. Zagorski, Austin J. Goodyke, Elizabeth A. VanSickle, Jeffrey P. Bond, Giselle L. Saulnier Sholler. The MDM2 inhibitor CGM097 synergizes with the BET inhibitor OTX015 to induce cell death in neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3191.

Objective For patients with end stage lung disease, lung transplantation (Ltx) is the unique treatment option. Nevertheless, a large number of lung allografts fail, because of acute or chronic rejection. Innate immune responses following Ltx play a critical role in the development of primary graft dysfunction, and ultimately promote the development of chronic lung allograft dysfunction, leading to increased mortality after lung transplantation. Here, we focus on the mechanisms responsible for disruption of the cells membranes integrity, resulting in the release of Damage Associated Molecular Patterns (DAMPs) and activation of cell death pathways triggering cell injury in lungs with different preservation status. Methods Rat lungs were exposed to cold ischemia (CI group, n=9) or warm ischemia (WI group, n=9). In CI group, after Perfadex flushing, and hypothermic preservation (4 h) left lung was transplanted. In WI group, after 1 h of in situ WI, flashing and 3 h cold preservation the left lung was transplanted. Lung mechanics were determined during reperfusion. Recipients were sacrificed at 30, 60 and 120 min after Ltx. Bronchoalveolar lavage (BAL) was performed on the grafts to measure markers of cellular injury, and cytokines. Cell death markers were determined in graft tissue. Biological samples were kept at -80°C for additional analysis. Results Compared to CI, WI group displayed significantly decreased graft function associated with increased activation of pyroptotic cell death (NLRP3, caspase1, IL-1β) pathway, whereas apoptotic (caspas3 and caspase7) and necroptotic (RIP1, RIP3, MLKL) cell death pathways activation were similar in both groups. WI group showed more important cellular damage with DAMPs release (LDH, sRAGE, HMGB) and inflammation (CXCL1, IL1-β, IL-33). Conclusion Our results suggest that pyroptosis could play a critical role in the development of early graft dysfunction in lungs subjected to warm ischemia. Inhibition of pyroptotic pathway could be a strategy to improve damaged donor lungs.

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

Pancreatic cancer exhibits the worst prognostic outcome among human cancers. Recently, we have described that depletion of RUNX2 enhances gemcitabine (GEM) sensitivity of p53-deficient pancreatic cancer AsPC-1 cells through the activation of TAp63-mediated cell death pathway. These findings raised a question whether RUNX2 silencing could also improve GEM efficacy on pancreatic cancer cells bearing p53 mutation. In the present study, we have extended our study to p53-mutated pancreatic cancer MiaPaCa-2 cells. Based on our current results, MiaPaCa-2 cells were much more resistant to GEM as compared with p53-proficient pancreatic cancer SW1990 cells, and there existed a clear inverse relationship between the expression levels of TAp73 and RUNX2 in response to GEM. Forced expression of TAp73α in MiaPaCa-2 cells significantly promoted cell cycle arrest and/or cell death, indicating that a large amount of TAp73 might induce cell death even in the presence of mutant p53. Consistent with this notion, overexpression of TAp73α stimulated luciferase activity driven by p53/TAp73-target gene promoters in MiaPaCa-2 cells. Similar to AsPC-1 cells, small interfering RNA-mediated knockdown of RUNX2 remarkably enhanced GEM sensitivity of MiPaCa-2 cells. Under our experimental conditions, TAp73 further accumulated in RUNX2-depleted MiaPaCa-2 cells exposed to GEM relative to GEM-treated non-silencing control cells. As expected, silencing of p73 reduced GEM sensitivity of MiPaCa-2 cells. Moreover, GEM-mediated Tyr phosphorylation level of TAp73 was much more elevated in RUNX2-depleted MiaPaCa-2 cells. Collectively, our present findings strongly suggest that knockdown of RUNX2 contributes to a prominent enhancement of GEM sensitivity of p53-mutated pancreatic cancer cells through the activation of TAp73-mediated cell death pathway, and also provides a promising strategy for the treatment of patients with pancreatic cancer bearing p53 mutation.

Pediatric Neuroblastoma (NB) is one of the most prevalent solid tumors in children that arise from embryonic neural crest cells and poses a significant challenge in the field of oncology. There are about 700 to 800 new cases of neuroblastoma diagnosed each year in the United States, and accounts for approximately 15% of all childhood cancer mortalities. Since this malignancy predominantly affects infants and young children, it is imperative to decipher the underlying molecular factors driving its pathogenesis. The novel molecular therapeutic approach includes the use of epigenetic modifiers as drugs. This approach aims to reverse significant epigenetic events underlying cancer pathogenesis, particularly abnormalities in histone modifications and DNA methylation. The reduction of both DNA and histone methylation levels leads to the reactivation of tumor suppressor genes and inhibits cancer cell proliferation and cell growth. In this study, we used selective and reversible small molecule agents such as MDM2 inhibitor (RG7388) and epigenetic modifiers (CM272 and SAHA) to block cell proliferation by inducing cell cycle arrest via enforcing Epigenetic alterations. Our initial experiments were designed to understand the effects of RG7388, CM272, and SAHA treatments on NB and their impact on cell cycle progression. It was hypothesized that the decrease in DNA methylation and increase in histone modification- augment p21 expression via the use of MDM2 inhibitors and epigenetic modifiers. To test this hypothesis, the NB cells (SK-N-SK and IMR-32) were treated with RG7388, CM272, and SAHA for 24 hours. The extent of Cell death was determined for NB cells by using the Trypan Blue Dye Exclusion (TBDE) method, and CellTiter-Glo® Luminescent Cell Viability Assay. Induction of apoptosis was determined by using the Immunofluorescence staining method with DEVD-amc substrate. The results of our study suggested that RG7388, CM272, and SAHA were able to induce cell death in the SK-N-SH cells following 24-hour treatment. Furthermore, we analyzed the levels of Tumor suppressor (p53), cell cycle arrest (p21, p27, AurkB, CDC25A, CyclinD) cell death (Bax, Bcl2, LC3, Beclin, PARP, Cleaved PARP, RIP1, RIP3, MLKL, p-AKT), and Epigenetic biomarker (DNMT1, DNMT3A, DNMT3B, acetyl histoneH3, H4, and H2b) protein expression levels using western blot analysis. The results showed significant elevation of p53, p21, p27, and acetylation of histone family biomarkers after treatment when compared with the control group. In summary, our results indicate that RG7388, CM272, and SAHA treatments can effectively induce cellular responses and prevent cell growth and progression by enforcing epigenetic changes in the NB cells. (This research was supported by the National Pediatric Cancer Foundation, Tampa, Florida, and the Royal Dames of Cancer Research Inc. of Ft. Lauderdale, Florida) Citation Format: Tahani Momenah, Umamaheswari Natarajan, Shyam Jaganathan, Appu Rathinavelu. Efficacy of MDM2 and epigenetic inhibitors treatments for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2865.

We recently learned that the cDNA encoding CEL purchased from OpenBiosystems (clone ID 5187959; GenBankTM accession no. BC042510.1) for this work contains a three-base pair in-frame deletion resulting in p.E365del. (The correct sequence should be 356NKGNKKVTEEDFYKLVSEFTITKGL380 and not 356NKGNKKVTE-DFYKLVSEFTITKGL380.) p.E365 is conserved in most primates. The residue is located in a surface loop of the CEL globular domain distant from the catalytic site and the bile acid-binding site. In our study, both CEL14R and CEL MODY contained p.E365del. The experimental variable was the sequence of the variable number of tandem repeat (VNTR) domain (Fig. 1A). Thus, the reported differences between CEL14R and CEL MODY reflect the effect of the VNTR MODY mutation on CEL folding. In support of this contention, another group that expressed a form of CEL with p.E365 also found that CEL MODY forms significant levels of intracellular protein aggregates compared with CEL14R (Johansson, B. B., Torsvik, J., Bjø;rkhaug, L., Vesterhus, M., Ragvin, A., Tjora, E., Fjeld, K., Hoem, D., Johansson, S., Ræder, H., Lindquist, S., Hernell, O., Cnop, M., Saraste, J., Flatmark, T., Molven, A., and Njø;lstad, P. R. (2011) Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): A protein misfolding disease. J. Biol. Chem. 286, 34593–34605). Because our hypothesis was that CEL MODY causes disease by misfolding and activating maladaptive cell signaling pathways, the presence of p.E365del raises concerns that deletion may have affected protein folding efficiency and contributed to the unfolded protein response we observed in our study. Based on our results with CEL14R, the effect of p.E365del on the folding of CEL appears minimal. CEL14R was robustly secreted and only formed low levels of detergent-insoluble intracellular aggregates (Figs. 2 and 5). CEL14R had good activity against triglycerides (Fig. 1) and was stimulated by bile acids (Xiao, X., and Lowe, M. E., unpublished observations). The results with CEL14R also showed that the presence of p.E365del was not sufficient to trigger activation of cell death and inflammatory pathways to the same level as seen with CEL MODY (Figs. 4 and 7). Still, we cannot exclude the possibility that the presence of p.E365del amplified the unfolded protein response elicited by the aggregation of CEL MODY resulting in a more robust activation of cell death pathways. Given this new information, the conclusion that “a mutation in CEL is sufficient to cause ER stress, induction of the UPR, NF-κB activation, and acinar cell death” in the first sentence of the last paragraph under “Discussion” is overstated. Even with this caveat, our data support the conclusion that disorders of protein homeostasis can have a role in the pathophysiology of chronic pancreatitis.

Mitochondria—Judges and Executioners of Cell Death Sentences

Editor's evaluation: Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates development and early survival of retinal neurons

Mitochondria play a central role in the initiation of apoptosis, which is regulated by various factors such as ATP synthesis, reactive oxygen species, redox status, and outer membrane permeabilization. Disruption of chicken thioredoxin 2 (Trx2), a mitochondrial redox-regulating protein, results in apoptosis in DT40 cells. To investigate the mechanism of this apoptosis, we prepared transfectants expressing control (DT40-TRX2-/-), human thioredoxin 2 (TRX2) (DT40-hTRX2), or redox-inactive TRX2 (DT40-hTRX2CS) in conditional Trx2-deficient DT40 cells containing a tetracycline-repressible Trx2 gene. Production of ATP was not significantly changed by down-regulation of Trx2 expression. The generation of reactive oxygen species was enhanced by the down-regulation of Trx2 expression in DT40-TRX2-/-. Unexpectedly, the change was blocked in both DT40-hTRX2 and DT40-hTRX2CS cells. The down-regulation of Trx2 expression caused the release of cytochrome c and apoptosis-inducing factor on day 3, and apoptosis on day 5. These changes were also suppressed in both DT40-hTRX2 and DT40-hTRX2CS cells, suggesting that TRX2 regulates mitochondrial outer membrane permeabilization and apoptosis by redox-active site cysteine-independent mechanisms. The down-regulation of Trx2 expression caused a decrease in the protein level of Bcl-xL on day 3, whereas the protein level of Bcl-2 did not change until day 4, and the mRNA level of Bcl-xL was unchanged. The decrease in Bcl-xL was not blocked by a caspase 3 inhibitor but blocked in both DT40-hTRX2 and DT40-hTRX2CS. These findings indicate a link between the redox active site cysteine-independent action of TRX2 and the level of Bcl-xL in the regulation of apoptosis.

Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.

Evidence for Mutant p53 Gain-of-Function Effects in Normal Haemopoietic Cells and Myc-Driven Lymphoma

The heat shock proteins (HSPs) are encoded by genes whose expression is substantially increased during stress conditions, such as heat shock, alcohol, inhibitors of energy metabolism, heavy metals, oxidative stress, fever or inflammation. During these conditions, HSPs increase cell survival by protecting and disaggregating stress‐labile proteins (Skowyra et al ., 1990), as well as the proteolysis of the damaged proteins (Wickner et al ., 1999). Under non‐stress conditions, HSPs have multiple housekeeping functions, such as folding and translocating newly synthesized proteins, activation of specific regulatory proteins, including transcription factors, replication proteins and kinases, protein degradation, protein signalling, including steroid hormone activation and tumour immunogenicity, and antigen presentation (for reviews see Helmbrecht et al ., 2000; Jolly and Morimoto, 2000). This broad spectrum of functions gave rise to the term ‘molecular chaperone’, an entity that acts to assist other proteins folding and maturating in the cell. It should also be emphasized that not all HSPs are molecular chaperones and not all chaperones are HSPs (Ellis and Hartl, 1999). HSPs are designated nomenclature according to their approximate molecular weight, e.g. the 70 kDa HSP is known as the molecular chaperone Hsp70. The 70 kDa heat shock‐related proteins comprise a family of highly conserved molecular chaperones that regulate a wide variety of cellular processes during normal and stress conditions (Boorstein et al ., 1994). Hsp70 is one of the most abundant of these proteins, accounting for as much as 1–2% of total cellular protein (Herendeen et al ., 1979). In humans, there are at least 11 distinct genes that code for Hsp70 isoforms, which are located on several different chromosomes (Tavaria et al ., 1996). The major, constitutively expressed hsp70 isoform is called hsc70 (gene product known as the clathrin‐uncoating ATPase or Hsp73) (Welch, 1992). The transcription of inducible forms of hsp70 or hsp72 are under …

Idasanutlin and Navitoclax Induce Synergistic Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

Outcome of NPM1 Mutated Acute Myeloid Leukemia (AML) Treated with Hypomethylating Agents (HMAs): A Report on 71 Cases