MDM2 Degradation Research Articles

Abstract 4515: Orally bioavailable PROTAC based MDM2 degrader

Abstract Activation of the tumor suppressor p53, through inhibition of the MDM2 protein, has been pursued as a cancer therapeutic strategy and has produced many distinct inhibitors that are in clinical trials. A limitation arising from inhibiting the MDM2-p53 interaction is the upregulation of MDM2, itself a target gene of p53, and this attenuates the activation of p53 and efficacy of the inhibitors. To circumvent this limitation, we previously reported the first PROTAC based MDM2 degraders. These induced and sustained robust degradation of MDM2, achieved stronger p53 activation, and more potent anticancer activity than MDM2 inhibitors thus representing a new therapeutic strategy for targeting MDM2. Currently, all the PROTAC based MDM2 degraders are all dosed intravenously. Here we describe our design, synthesis, and optimizations that led to the discovery of the first orally bioavailable PROTAC based MDM2 degrader and our investigation of its therapeutic potential and mechanism of action. Consistent with its design to effectively degrade MDM2, our PROTAC effectively induces rapid degradation of MDM2 resulting in accumulation of wild-type p53 protein and activates p53 transcriptional activity in leukemia cells without accumulation of MDM2 protein. Consequently, it potently inhibits cell growth and induces apoptosis at low nano-molar concentrations in ALL and AML cell lines >10-100 times more potent than MDM2 inhibitors; and, after 50 mg/kg oral dose, 5 days/week for 3 weeks, it increased the median survival by 26 days of mice with disseminated RS4;11 cancer. Citation Format: Angelo Aguilar, Jiuling Yang, Yangbing Li, Donna McEachern, Shaomeng Wang. Orally bioavailable PROTAC based MDM2 degrader [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 4515.

MDM2 Inhibitors for Cancer Therapy: The Past, Present, and Future.

Since its discovery over 35 years ago, MDM2 has emerged as an attractive target for the development of cancer therapy. MDM2's activities extend from carcinogenesis to immunity to the response to various cancer therapies. Since the report of the first MDM2 inhibitor more than 30 years ago, various approaches to inhibit MDM2 have been attempted, with hundreds of small-molecule inhibitors evaluated in preclinical studies and numerous molecules tested in clinical trials. Although many MDM2 inhibitors and degraders have been evaluated in clinical trials, there is currently no Food and Drug Administration (FDA)-approved MDM2 inhibitor on the market. Nevertheless, there are several current clinical trials of promising agents that may overcome the past failures, including agents granted FDA orphan drug or fast-track status. We herein summarize the research efforts to discover and develop MDM2 inhibitors, focusing on those that induce MDM2 degradation and exert anticancer activity, regardless of the p53 status of the cancer. We also describe how preclinical and clinical investigations have moved toward combining MDM2 inhibitors with other agents, including immune checkpoint inhibitors. Finally, we discuss the current challenges and future directions to accelerate the clinical application of MDM2 inhibitors. In conclusion, targeting MDM2 remains a promising treatment approach, and targeting MDM2 for protein degradation represents a novel strategy to downregulate MDM2 without the side effects of the existing agents blocking p53-MDM2 binding. Additional preclinical and clinical investigations are needed to finally realize the full potential of MDM2 inhibition in treating cancer and other chronic diseases where MDM2 has been implicated. SIGNIFICANCE STATEMENT: Overexpression/amplification of the MDM2 oncogene has been detected in various human cancers and is associated with disease progression, treatment resistance, and poor patient outcomes. This article reviews the previous, current, and emerging MDM2-targeted therapies and summarizes the preclinical and clinical studies combining MDM2 inhibitors with chemotherapy and immunotherapy regimens. The findings of these contemporary studies may lead to safer and more effective treatments for patients with cancers overexpressing MDM2.

Translating p53-based therapies for cancer into the clinic.

Inactivation of the most important tumour suppressor gene TP53 occurs in most, if not all, human cancers. Loss of functional wild-type p53 is achieved via two main mechanisms: mutation of the gene leading to an absence of tumour suppressor activity and, in some cases, gain-of-oncogenic function; or inhibition of the wild-type p53 protein mediated by overexpression of its negative regulators MDM2 and MDMX. Because of its high potency as a tumour suppressor and the dependence of at least some established tumours on its inactivation, p53 appears to be a highly attractive target for the development of new anticancer drugs. However, p53 is a transcription factor and therefore has long been considered undruggable. Nevertheless, several innovative strategies have been pursued for targeting dysfunctional p53 for cancer treatment. In mutant p53-expressing tumours, the predominant strategy is to restore tumour suppressor function with compounds acting either in a generic manner or otherwise selective for one or a few specific p53 mutations. In addition, approaches to deplete mutant p53 or to target vulnerabilities created by mutant p53 expression are currently under development. In wild-type p53 tumours, the major approach is to protect p53 from the actions of MDM2 and MDMX by targeting these negative regulators with inhibitors. Although the results of at least some clinical trials of MDM2 inhibitors and mutant p53-restoring compounds are promising, none of the agents has yet been approved by the FDA. Alternative strategies, based on a better understanding of p53 biology, the mechanisms of action of compounds and treatment regimens as well as the development of new technologies are gaining interest, such as proteolysis-targeting chimeras for MDM2 degradation. Other approaches are taking advantage of the progress made in immune-based therapies for cancer. In this Review, we present these ongoing clinical trials and emerging approaches to re-evaluate the current state of knowledge of p53-based therapies for cancer.

Anti-Leukemic Effects of CK1α Degrader As Monotherapy and in Combination with Targeted Drugs

CK1⍺ is a member of casein kinase 1 (CK1) family, a multifunctional serine/threonine kinase conserved in eukaryotes from yeast to humans. Substrates of CK1⍺ include various proteins important in cancer and regulate multiple survival pathways such as p53, Wnt, autophagy and NF-B signaling pathways. In particular, CK1- serves as an upstream regulator of p53 pathway through regulation of MDM2 and MDMX and degradation of CK1- may prevent MDM2 and/or MDMX mediated inactivation of p53 function, thus facilitate cell death. We identified an early hit compound, distinct from known immunomodulatory imide drugs (IMiDs) such as lenalidomide or thalidomide. In order to identify compounds having distinct characteristic with previously published IMiDs, we designed multiple compounds with a wide range of glue effect based on the binding mode analysis and binding structure of CRBN and known IMiDs. Among them, PinA1 (early lead compound) selectively degraded CK1-, in the nanomolar range with the highest potency. PinA1 induced expression of p53 which led to apoptosis in AML cell lines with wild-type TP53 (MV4-11, MOLM-13, and MOLM-14), but not in TP53 mutated cells (KASUMI-1, and KG-1). Of note, in TP53 wild-type cells, expression of MDM2 and p21 increased as well, which may limit p53-induced apoptosis in these cells. PinA1 also induced CK1- degradation and p53-dependent apoptosis in primary AML cells in vitro. On the other hand, PinA1 had marginal effects on the viability of human peripheral blood mononuclear cells and CD34+ cells, with minimal activation of the p53 pathway at an efficacious dose range for MV4-11 compared to MDM2 inhibitors. PinA1 enhanced apoptosis in combination with FLT3, BCL-2, or MDM2 inhibitors in AML cell lines (MV4-11, MOLM-14) and primary AML cells in vitro. To test the efficacy of PinA1 in vivo, we injected MOLM-14/ luc/GFP or MV4-11/ luc/Thy1.1 into nonirradiated NGS mice. Five or Six days after cell injection, mice were randomized into 4 groups: vehicle, PinA1, and PinA1 plus AMG232 (MDM2 inhibitor), venetoclax, or gilteritinib. Bioluminescence imaging (BLI) demonstrated significantly less leukemia burden in all treated groups than vehicles; leukemia progression was lowest with combination therapy. Flow cytometry with sacrificed mice revealed the lowest leukemia burden in combination therapy, consistent with those from BLI. Western blotting with sorted leukemic cells from bone marrow and spleen revealed CK1- degradation in vivo. PinA1- and other single drug-treated mice had prolonged survival compared with vehicles, and combination therapy extended survival even further. Patient-derived xenograft models from two FLT3-ITD-mutated AML patients also showed potent anti-leukemic effects of PinA1 as monotherapy and in combination with gilteritinib. In conclusion, our study showed that PinA1 selectively degrades CK1α, enhancing expression of p53, which then induces apoptosis in TP53-wild-type AML cells, but not in TP53-mutated AML cells, thereby producing anti-leukemic effects as monotherapy or in combination with targeted therapies. The minimal effect of PinA1 on normal hematopoietic cells may positively contribute to safety in future clinical trials. This novel targeting approach may improve the current targeted therapies in combination once validated.

USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

USP13 regulates cell senescence through mediating MDM2 stability

AimsLung aging results in altered lung function, reduced lung remodeling and regenerative capacity, and increased susceptibility to acute and chronic lung diseases. The molecular and physiological underlying mechanisms of lung aging remain unclear. Mounting evidence suggests that deubiquitinating enzymes (DUBs) play a critical role in tissue aging and diseases through regulation of cellular signaling pathways. Here we investigate the role of Ubiquitin-Specific Protease 13 (USP13) in cell senescence and lung aging and its underlying mechanisms. Main methodsProtein levels of USP13 and MDM2 in lung tissues from aged and young mice were compared. Gene silencing and overexpression of USP13 in human cell lines were performed. MDM2 levels were examined by Quantitative Real-Time PCR and Western blotting analysis. The cell senescence levels of human cells were checked by the β-galactosidase staining. Key findingsLung tissues from aged mice showed higher levels of USP13 compared to younger mice. We found a negative correlation between USP13 and MDM2 expression in lung tissues of aged mice. The increased protein levels of MDM2 were detected in lung tissues of USP13 deficient mice. Furthermore, overexpression of USP13 promoted cell senescence. Knockdown of USP13 increased MDM2 levels in lung cells, while overexpression of USP13 reduced it. The degradation of MDM2 caused by USP13 was prevented by the proteasome inhibitor MG132. Furthermore, we showed that USP13 targeted and reduced K63-linked polyubiquitination of MDM2. These results demonstrate that USP13 is involved in the aging signaling pathway in lungs through regulation of MDM2.

Correction: Targeted MDM2 Degradation Reveals a New Vulnerability for p53-Inactivated Triple-Negative Breast Cancer

Correction: Targeted MDM2 Degradation Reveals a New Vulnerability for p53-Inactivated Triple-Negative Breast Cancer

Abstract 2784: JW-1-283 inhibits melanoma tumor growth via the stabilization of p53 pathway

Abstract Melanoma is the most lethal skin cancer, with the increasing incidence in the past decade. Despite the significant advancements in immune therapy and targeted therapy, the overall prognosis for metastatic melanoma remains unsatisfactory. TP53 is a tumor suppressor, but its wildtype is inactivated in about 90% of melanoma. Since activated p53 can promote cancer cell apoptosis and thus effectively inhibit tumor growth and tumor metastasis, reactivation of p53 is considered a promising strategy for cancer therapy. Herein, we report a recently developed small molecule compound, JW-1-283, targeting the interactions between MDM2 E3 ubiquitin-protein ligase and p53 protein. In vitro, JW-1-283 showed potent cytotoxicity against A375 melanoma cell line (IC50 2.2±0.3 μM) which has wildtype p53, but its potency is substantially reduced in two other melanoma cell lines with either p53 mutation (M14) or p53 null (RPMI-7951) status. Treatment with JW-1-283 significantly dose-dependently reduced the stemness in A375 melanoma cells, as indicated by low numbers of colony formation, impaired tumor sphere formation abilities, and inhibition of cancer cell migration. Mechanistically, JW-1-283 disrupts the p53-MDM2 interaction, prevents p53 and MDM2 degradation in the existence of cycloheximide, and induces p38 and p53 phosphorylation in the apoptotic pathway. Genetic knockdown of p38 by siRNA further confirmed that the stabilized p53 could be subsequently phosphorylated by p38, leading to induced apoptosis as well as S phase cell cycle arrest. In vivo, when A375 tumors grown in NSG mice were treated with 7.5 mg/kg of JW-1-283 intraperitoneally every other day for a consecutive of 16 days, significant inhibition of tumor growth was observed, along with decreased tumor cell proliferation, tumor angiogenesis, and enhanced tumor cell apoptosis. In summary, this work provides the initial proof-of-concept of developing the JW-1-283 scaffold as a potential strategy for advanced melanoma, which inhibits tumor growth by activating the p53 signaling pathway. Citation Format: Yang Xie, Kelli Hartman, Zhongzhi Wu, Wei Li. JW-1-283 inhibits melanoma tumor growth via the stabilization of p53 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2784.

Targeted MDM2 Degradation Reveals a New Vulnerability for p53-Inactivated Triple-Negative Breast Cancer.

p53-inactivated TNBC is an aggressive, therapy-resistant, and lethal breast cancer subtype. We designed a new compound targeting an unexpected vulnerability we identified in TNBC. Our MDM2-targeted degrader kills p53-inactivated TNBC cells, highlighting the requirement for MDM2 in TNBC cell survival and as a new therapeutic target for this disease. See related commentary by Peuget and Selivanova, p. 1043. This article is highlighted in the In This Issue feature, p. 1027.

Design, synthesis, and biological evaluation of novel protopanoxadiol derivatives based PROTACs technology for the treatment of lung cancer

Protopanoxadiol is a key active ingredient derived from Panax ginseng that is well-known to exhibit anti-tumor activity. Previous research focused on the natural protopanaxadiol derivative AD-1 has demonstrated that it possesses broad spectrum anti-tumor activities in vitro and in vivo. However, its limited activity, selectivity, and cell permeability have impeded its therapeutic application. Herein, a series of novel AD-1 derivatives were designed and synthesized based on proteolysis-targeting chimera (PROTAC) technology by linking AD-1 at the C-3 and C-12 positions with pomalidomide through linkers of alkyl chain of differing lengths to achieve the goal of improving the efficacy of the parent compound. Among these synthesized PROTACs, the representative compound A05 exhibited the most potent anti-proliferative activity against A549 cells. Furthermore, mechanistic studies revealed that compound A05 was able to suppress MDM2 expression, disrupt interactions between p53 and MDM2 and readily induce apoptotic death via the mitochondrial apoptosis pathway. Moreover, the in vivo assays revealed that compound A05 exhibited both anti-proliferative and anti-metastatic activities in the zebrafish tumor xenograft model with A549 cells. Together, our findings suggest that AD-1 based PROTACs associated with the degradation of MDM2 may have promising effects for the treatment of lung cancer and this work provide a foundation for future efforts to develop novel anti-tumor agents from natural products.

An MDM2 degrader for treatment of acute leukemias.

In acute myeloid leukemia (AML), p53 tumor suppressor activity can be reduced due to enhanced expression of MDM2 which promotes the degradation of p53. In TP53 wild-type malignancies, therapy with small molecule antagonists of MDM2 results in antileukemic activity. Current treatment strategies, however, have been limited by poor tolerability and incomplete clinical activity. We have developed a proteolysis-targeting chimera (PROTAC) MS3227 that targets MDM2 by recruiting the E3 ligase Von Hippel-Lindau, resulting in proteasome-dependent degradation of MDM2. In WT TP53 leukemia cell lines, MS3227 led to activation of p53 targets p21, PUMA, and MDM2 and resulted in cell-cycle arrest, apoptosis, and decreased viability. The catalytic PROTAC MS3227 led to more potent activation when compared to a stoichiometric inhibitor, in part by dampening the negative feedback mechanism in the p53 - MDM2 circuit. The effectiveness of MS3227 was also observed in primary patient specimens with selectivity towards leukemic blasts. The addition of MS3227 enhanced the activity of other anti-leukemic agents including azacytidine, cytarabine, and venetoclax. In particular, MS3227 treatment was shown to downregulate MCL-1, a known mediator of resistance to venetoclax. A PROTAC-based approach may provide a means of improving MDM2 inhibition to gain greater therapeutic potential in AML.

Design, synthesis and biological evaluation of 2-aminopyridine derivatives as USP7 inhibitors

A series of novel 2-aminopyridine derivatives 1–26 have been designed and synthesized by structural modifications on a lead USP7 inhibitor, GNE6640. All the compounds were evaluated for their USP7 inhibitory activities. The results showed that most of the compounds have good USP7 inhibitory activities at the concentration of 50 μM. Among them, compounds 7, 14 and 21 are the most potential ones from each category with the IC50 values of 7.6 ± 0.1 μM, 17.0 ± 0.2 μM and 11.6 ± 0.5 μM, respectively. Compounds 7 and 21 expressed significant binding interactions with USP7 by surface plasmon resonance (SPR)-based binding assay, but both of them presented moderate antiproliferative activities against HCT116 cells. They could effectively promote MDM2 degradation, p53 stabilization and p21 gene expression in the western blot analysis.

Upregulation of p53 through induction of MDM2 degradation: improved potency through the introduction of an alkylketone sidechain on the anthraquinone core

Overexpression of ubiquitin ligase MDM2 causes depletion of the p53 tumour-suppressor and thus leads to cancer progression. In recent years, anthraquinone analogs have received significant attention due to their ability to downregulate MDM2, thereby promoting p53-induced apoptosis. Previously, we have developed potent anthraquinone compounds having the ability to upregulate p53 via inhibition of MDM2 in both cell culture and animal models of acute lymphocytic leukaemia. Earlier work was focussed on mechanistic work, pharmacological validation of this class of compounds in animal models, and mapping out structural space that allows for further modification and optimisation. Herein, we describe our work in optimising the substituents on the two phenol hydroxyl groups. It was found that the introduction of an alkylketone moiety led to a potent series of analogs with BW-AQ-350 being the most potent compound yet (IC50 = 0.19 ± 0.01 µM) which exerts cytotoxicity by inducing MDM2 degradation and p53 upregulation.

Ubiquitin receptors play redundant roles in the proteasomal degradation of the p53 repressor MDM2.

Much remains to be determined about the participation of ubiquitin receptors in proteasomal degradation and their potential as therapeutic targets. Suppression of the ubiquitin receptor S5A/PSMD4/hRpn10 alone stabilises p53/TP53 but not the key p53 repressor MDM2. Here, we observed S5A and the ubiquitin receptors ADRM1/PSMD16/hRpn13 and RAD23A and B functionally overlap in MDM2 degradation. We provide further evidence that degradation of only a subset of ubiquitinated proteins is sensitive to S5A knockdown because ubiquitin receptor redundancy is commonplace. p53 can be upregulated by S5A modulation while degradation of substrates with redundant receptors is maintained. Our observations and analysis of Cancer Dependency Map (DepMap) screens show S5A depletion/loss substantially reduces cancer cell line viability. This and selective S5A dependency of proteasomal substrates make S5A a target of interest for cancer therapy.

Functions and roles of IFIX, a member of the human HIN-200 family, in human diseases.

Pyrin and hematopoietic expression, interferon-inducible nature, and nuclear localization (HIN) domain family member 1 (PYHIN1), also known as IFIX, belongs to the family of pyrin proteins. This family includes structurally and functionally related mouse (e.g., p202, p203, and p204 proteins) and human (e.g., the interferon-inducible protein 16, absent in melanoma 2 protein, myeloid cell nuclear differentiation antigen, and pyrin and HIN domain family 1 or IFIX) proteins. The IFIX protein belongs to the HIN-200 family of interferon-inducible proteins that have a 200-amino acid signature motif at their C-termini. The increased expression of pyrin proteins in most cell types inhibits cell cycle control and modulates cell survival. Consistent with this role for pyrin proteins, IFIX is a potential antiviral DNA sensor that is essential for immune responses, the detection of viral DNA in the nucleus and cytoplasm, and the binding of foreign DNA via its HIN domain in a sequence non-specific manner. By promoting the ubiquitination and subsequent degradation of MDM2, IFIX acts as a tumor suppressor, thereby leading to p53/TP53 stabilization, HDAC1 regulation via the ubiquitin-proteasome pathway, and tumor-cell-specific silencing of the maspin gene. These data demonstrate that the potential molecular mechanism(s) underlying the action of the IFIX protein might be associated with the development of human diseases, such as viral infections, malignant tumors, and autoimmune diseases. This review summarizes the current insights into IFIX functions and how its regulation affects the outcomes of various human diseases.

Design of stapled peptide-based PROTACs for MDM2/MDMX atypical degradation and tumor suppression.

Rationale: Although stapled peptides offer a powerful solution to overcome the susceptibility of linear peptides to proteolytic degradation and improve their ability to cross membranes, an efficient and durable disease treatment strategy has not yet been developed due to the inevitable elimination of peptide inhibitors and rapid accumulation of target proteins.Methods: Herein we developed stapled peptide-based proteolysis-targeting chimeras (SP-PROTACs), that simultaneously exhibited improved cellular uptake and proteolytic stability attributed to the stapled peptides, and efficient target protein degradation promoted by the PROTACs. Based on the PMI peptide with dual specificity for both MDM2 and MDMX, a series of SP-PROTACs were designed.Results: Among them, the optimized SPMI-HIF2-1 exhibited similar binding affinity with MDM2 and MDMX but obviously higher helical contents, improved proteolytic stability, better cellular permeability, and a better pharmacokinetic profile compared with its linear counterpart. Importantly, SPMI-HIF2-1 could effectively kill cancer cells and inhibit tumor progression in subcutaneous and orthotopic colorectal cancer xenograft models through simultaneously promoting the atypical degradation of both MDM2 and MDMX and durable p53 activation. An FP-based binding assay and structural modeling analysis of the ternary complex suggested that SPMI-HIF2-1 simultaneously bound with the target protein and E3 ligase.Conclusion: Our findings not only provide a new class of anticancer drug candidates, but also bridge the gap and reduce the physical distance between peptides and PROTACs.

Stability and bifurcation analyses of p53 gene regulatory network with time delay

<abstract><p>In this paper, based on a p53 gene regulatory network regulated by Programmed Cell Death 5(PDCD5), a time delay in transcription and translation of Mdm2 gene expression is introduced into the network, the effects of the time delay on oscillation dynamics of p53 are investigated through stability and bifurcation analyses. The local stability of the positive equilibrium in the network is proved through analyzing the characteristic values of the corresponding linearized systems, which give the conditions on undergoing Hopf bifurcation without and with time delay, respectively. The theoretical results are verified through numerical simulations of time series, characteristic values and potential landscapes. Furthermore, combined effect of time delay and several typical parameters in the network on oscillation dynamics of p53 are explored through two-parameter bifurcation diagrams. The results show p53 reaches a lower stable steady state under smaller PDCD5 level, the production rates of p53 and Mdm2 while reaches a higher stable steady state under these larger ones. But the case is the opposite for the degradation rate of p53. Specially, p53 oscillates at a smaller Mdm2 degradation rate, but a larger one makes p53 reach a low stable steady state. Besides, moderate time delay can make the steady state switch from stable to unstable and induce p53 oscillation for moderate value of these parameters. Theses results reveal that time delay has a significant impact on p53 oscillation and may provide a useful insight into developing anti-cancer therapy.</p></abstract>

The Inhibitory Effects of 6-Thioguanine and 6-Mercaptopurine on the USP2a Target Fatty Acid Synthase in Human Submaxillary Carcinoma Cells

Overexpression of the deubiquitinase USP2a leads to stabilization of fatty acid synthase (FAS), the levels of which are often elevated in aggressive human cancers. Consequently, there is an urgent need for inhibitors to suppress the deubiquitination activity of USP2a so as to upregulate FAS protein degradation. We first analyzed the relationship between the expression level of USP2a and survival using The Cancer Genome Atlas Head-Neck Squamous Cell Carcinoma (HNSC) data collection. Our results suggested survival rates were lower among HNSC patients expressing higher levels of USP2a. We then investigated two thiopurine drugs, 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP), to determine whether they could potentially serve as inhibitors of USP2a. Western blot analysis showed that levels of two USP2a target proteins, FAS and Mdm2, were dose-dependently decreased in A253 submaxillary carcinoma cells treated with 6-TG or 6‐MP. Responding to the degradation of Mdm2, levels of p53 were increased. We found that 6-TG and 6-MP also suppressed levels of both USP2a mRNA and protein, suggesting these two thiopurines do not act solely through direct inhibition of USP2a. The effects of 6-TG and 6-MP were not cell type-specific, as they elicited similar decreases in FAS protein in leukemia, prostate and cervical cancer cell lines. 6-TG and 6-MP had effects on several cell cycle proteins, including another USP2a target protein, cyclin D1. The populations of cells in subG1 and S phase were increased by 6-TG and 6-MP, which was accompanied by reductions in G1 phase cells. In untreated cells, USP2a transfection increased FAS and cyclin D1 levels compared to an enzyme-dead USP2a C276A mutant, which lacked deubiquitinating activity. However, USP2a transfection failed to reverse the suppressive effects of 6‐TG and 6-MP on FAS levels. In summary, these findings suggest 6-TG and 6-MP reduce the stability of some USP2a targets, including FAS and Mdm2, by inhibiting USP2a-catalyzed deubiquitination in some cancer cells. Our work also provides repurposing evidence supporting 6‐TG and 6-MP as target therapeutic drugs, such as USP2a/FAS in this study.

Development of an MDM2 Degrader for Treatment of Acute Leukemias

IntroductionAcute myeloid leukemias (AMLs) are characterized by suppressed cell death pathways which promote leukemic blast survival. TP53 acts as a tumor suppressor gene in AML and is found mutated or deleted in 10-15% of patients. In a majority of cases though, TP53 is wild-type. Other mechanisms including MDM2 over-expression lead to reduced TP53 activity. MDM2 acts as a negative regulator by direct binding of TP53 and mediating TP53 degradation through ubiquitination. MDM2 itself is a transcriptional target of TP53 as a negative feedback mechanism limiting the function of TP53. Small molecule inhibition of MDM2 , blocking its ability to bind TP53, can activate TP53 and trigger cell cycle arrest and apoptosis through increased transcription of TP53 target genes. Increased MDM2 expression has been observed in hematologic malignancies including AML, providing rationale for clinical trials with MDM2 inhibitors. These agents such as RG7388 and AMG232 have shown efficacy as monotherapy and in combination. However, these agents have also exhibited toxicity and have yet to demonstrate sufficient benefit for their approval. To create more effective agents against MDM2, we have developed an MDM2 degrader XY-27 that functions as a proteolysis-targeting chimera (PROTAC). Based on relatively higher expression in AML compared to other cancer types, we selected VHL as the E3 ubiquitin ligase target for XY-27 , as this may improve specificity and potency in AML.ResultsThe PROTAC degrader XY-27 concurrently binds MDM2 and VHL, and by bringing these targets in proximity, VHL can then ubiquitinate MDM2, leading to its degradation by the proteasome. XY-27 can mediate degradation of MDM2 in a concentration dependent manner in the U937 leukemia cell line (Fig 1a). MDM2 degradation with XY-27 is blocked by proteasome inhibition and competitive binding of the VHL ligand. A control compound, which only differs in that it cannot bind to VHL, lacks degrader activity. Although MDM2 is itself an E3 ligase, VHL expression is not appreciably changed with XY-27 (Fig 1a).Treatment with XY-27 leads to apoptosis and decreased proliferation of leukemia cell lines in a TP53 dependent manner. Inhibition of MDM2 leads to up-regulation of TP53 and in TP53 wild-type cells, downstream targets CDKN1A (p21) and PUMA. MDM2 is also up-regulated through a feedback mechanism. XY-27 demonstrated greater potency than the MDM2-binding inhibitor AMG232 in the MOLM13 and MV4-11 leukemia cell lines (Fig 1b). Treatment with XY-27 led to higher levels of TP53 and p21 protein than with AMG232. CRISPR-mediated knock-out of VHL leads to reduced XY-27 potency. XY-27 also shows efficacy when combined with other chemotherapeutic agents such as azacytidine and cytarabine. In a long-term co-culture model with an OP9 feeder layer, XY-27 was capable of inducing apoptosis in primary patient AML samples (Fig 1c).ConclusionWe describe a new MDM2 PROTAC, XY-27 that demonstrates TP53 dependent activity against leukemia cells. It also demonstrates increase potency compared to an MDM2 binding inhibitor. Utilization of the PROTAC system has potential advantages through selection of the VHL E3 ubiquitin ligase. Because of negative feedback mechanisms involving TP53 and MDM2, direct binding inhibitors of MDM2 may be limited in activity through continued accumulation of MDM2. PROTAC degraders have catalytic activity and may overcome this inhibition by continued degradation of the target MDM2, and thus achieve greater TP53 activity.Figure 1. Activity of the MDM2-PROTAC XY-27 in leukemia. (a) Western blot from treatment of U937 leukemia cells with XY-27 for 24 hrs, at various concentrations (5 nM to 1 μM), resulting in the degradation of MDM2. (b) Dose response curves from treatment of MOLM13 and MV4-11 cell lines with XY-27 (blue) and AMG232 (red) for 48 hrs, demonstrating greater potency of XY-27. (c) Induction of apoptosis in primary AML cells treated with XY-27 at 1μM using a co-culture system for 3 days. *p<.05 [Display omitted] DisclosuresHoffman: Protagonist Therapeutics, Inc.: Consultancy; AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding; Novartis: Other: Data Safety Monitoring Board, Research Funding; Kartos Therapeutics, Inc.: Research Funding.

SETD2 Non-Genomic Loss of Function and p53 Functional Inactivation in Advanced Systemic Mastocytosis (SM): Pathogenetic and Therapeutic Implications

KIT constitutive activation is thought to be necessary but not sufficient for the development of aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). The identification and dissection of cooperating pathogenetic pathways may set the basis for the development of novel therapeutic strategies.We have recently found that the HMC-1.1 and -1.2 MCL cell lines and ASM/MCL patients (pts) display histone H3 Lys36 trimethylation (H3K36Me3) deficiency as a result of non-genomic loss of function of the SETD2 methyltransferase. SETD2 plays a key role in the control of transcription and splicing fidelity, homologous recombination (HR), mismatch repair (MMR). Inhibition of proteasome-mediated degradation by bortezomib restored SETD2 protein expression and H3K36Me3. Reversing the detrimental effects of SETD2 and H36K36Me3 deficiency may be a promising therapeutic strategy.In this study, we aimed to investigate the effects of SETD2 loss of function in ASM and MCL and to assess the in vitro efficacy of second-generation proteasome inhibitors as therapeutic agents. To investigate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting and immunofluorescence to assess phosphorylated histone 2A.X (γH2AX), which marks ongoing DNA damage signaling and DNA double strand breaks and Rad51, which is a surrogate for active HR. Compared to cells from healthy controls, cells from SETD2- and H3K36Me3-deficient MCL pts had significantly higher levels of γH2AX and Rad51. To assess whether the increased DNA damage may translate into a greater number of submicroscopic genomic lesions, and whether the degree of SETD2 down-modulation may be inversely correlated with the degree of genomic complexity, SNP-array data from 13 pts were examined. Structural complexity score (SCS) and genome instability index (GII) were not significantly different (SCS, 387 vs 376; GII, 0.293 vs 0.337) between pts with > or <50% residual SETD2 and H3K36Me3 levels, suggesting that the activity of HR repair may counteract DNA damage events. Moreover, SETD2 down-modulation was not associated with reduced MMR proficiency, as assessed by lack of microsatellite instability. On the other hand, RNA-seq in SETD2-deficient pts showed evidence of transcription and splicing defects like transcription-induced chimeras and intron retention not observed in healthy donors.In renal cell carcinoma cell lines, SETD2 has been shown to bind and activate p53. After proteasome inhibition, SETD2 was indeed found to immunoprecipitate with p53 in HMC-1.1 and -1.2 cells. Rescuing the interaction between SETD2 and p53 with bortezomib stabilized p53 and upregulated the expression levels of p53 targets including p21, p27, Bax and Gadd45a. This restored inhibition of cell proliferation, block of the transition towards cell cycle checkpoints and consequent activation of apoptosis. The ubiquitin E3 ligase MDM2 was also found to complex with SETD2 after proteasomal inhibition. Treatment with SP-141, a new class of MDM2 inhibitors that promote MDM2 auto-ubiquitination and degradation, also rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP-141 treatment of HMC-1 cells at micromolar doses for 24-48-72-96 hours induced cytostatic but not cytotoxic effects as shown by cell growth curves. Cytostatic effects resulted from a G2/M block of cell cycle induced by Gadd45a overexpression. Clonogenic assays supported the cytostatic effects of SP141 in HMC-1.1 and -1.2 cells (LD50=0.133µM and 0.050µM respectively).Finally, treatment with the second-generation proteasome inhibitors carfilzomib and ixazomib potently induced apoptosis and reduced colony growth in HMC-1.1 (LD50=0.10nM and 0.66nM respectively) and -1.2 (LD50=0.13nM and 0.72nM respectively) cells.In conclusion: MDM2-mediated ubiquitination contributes to SETD2 non-genomic loss of function in ASM and MCL. Loss of SETD2 and H3K36Me3 is associated with increased DNA damage and transcription and splicing defects in ASM and MCL pts and is likely to afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for TP53 gene mutations. Approaches aimed to inhibit MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies worth to be further explored.Supported by AIL and AIRC (16996) DisclosuresZanotti:Deciphera: Consultancy. Ciceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Valent:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; Ariad: Honoraria, Research Funding; Incyte: Honoraria; Deciphera: Honoraria, Research Funding; Pfizer: Honoraria; Blueprint: Research Funding; Teva: Honoraria. Martinelli:Amgen: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Roche: Consultancy; Johnson&Johnson: Consultancy; Ariad/Incyte: Consultancy. Soverini:Incyte Biosciences: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy.