Y220C Mutation Research Articles

TP53 Y220C mutations in pancreatic ductal adenocarcinoma (PDAC): A genomic landscape study.

761 Background: A novel targeted therapy designed to restore wild-type function to the p53 Y220C mutant protein has demonstrated promising activity in an early-phase trial (NCT04585750). While the TP53 Y220C mutation is found in < 1% of all solid malignancies, its prevalence is enriched in certain cancers including PDAC. Here we report results of comprehensive genomic profiling (CGP) in a cohort of patients with advanced PDAC, with a focus on TP53 Y220C mutations. Methods: A total of 27,377 cases of clinically advanced PDAC underwent hybrid capture-based CGP to assess all classes of genomic alterations (GAs). Cases were sequenced to a mean coverage depth of 650X. Microsatellite instability (MSI) status and tumor mutation burden (TMB) were determined using sequencing data; PD-L1 expression was measured by immunohistochemistry (Dako 22C3, tumor proportion score [TPS]). Results: TP53 Y220C alterations were identified in 488 (1.8%) PDAC patients (Y220C+). In comparison to PDAC patients lacking the TP53 Y220C alteration (Y220C-), Y220C+ patients were of similar age, but were more likely to be female (52.9% vs 46.8%; p = 0.0008) and harbor KRAS mutations (96.7% vs 92.7%; p = 0.007). Meanwhile, alterations in ATM were more frequent in the Y220C- group (4.0% vs 1.4%; p = 0.0008). Frequencies of other GAs were similar between Y220C+ and Y220C- groups, including alterations in the homologous recombination defect-associated genes BRCA1/2 and RAD21 , as well as ERBB2 , BRAF , MTAP and PIK3CA . MSI-high status and elevated TMB (≥ 10 mutations/Mb) were extremely uncommon in both groups. PD-L1 expression was similar in the Y220C+ and Y220C- groups, with positive PD-L1 expression (TPS ≥ 1) observed in 31.5% and 34.9% of patients, respectively. Conclusions: The potentially targetable TP53 Y220C mutation was identified in close to 2% of clinically advanced PDAC cases. Those with TP53 Y220C mutations were more likely to be female and harbor KRAS mutations, and less likely to harbor ATM mutations. However, TP53 Y220C mutations were not associated with other distinct genomic characteristics. These findings are consistent with previous reports of interactions between TP53 and KRAS alterations in PDAC. Given the emergence of drugs aimed at restoring p53 function in cases with the Y220C mutation, further study of this alteration in PDAC is warranted. Age, number of genomic alterations (GA) per tumor, and alteration frequency of select commonly altered genes based on TP53 Y220C status. TP53 Y220C+ (n = 488) TP53 Y220C- (n = 26,889) P Value Median Age, years (range) 66 (36-89+) 66 (21-89+) NS GA/tumor 5.2 5.0 NS KRAS 96.7% 92.7% 0.007 CDKN2A 60.1% 56.6% NS CDKN2B 28.6% 29.5% NS MTAP 24.4% 23.8% NS ARID1A 7.8% 8.7% NS ATM 1.4% 4.0% 0.0008 BRCA2 2.3% 3.1% NS NS = non-significant.

Discovery of Rezatapopt (PC14586), a First-in-Class, Small-Molecule Reactivator of p53 Y220C Mutant in Development.

p53 is a potent transcription factor that is crucial in regulating cellular responses to stress. Mutations in the TP53 gene are found in >50% of human cancers, predominantly occurring in the DNA-binding domain (amino acids 94-292). The Y220C mutation accounts for 1.8% of all of the TP53 mutations and produces a thermally unstable protein. Rezatapopt (also known as PC14586) is the first small-molecule p53 Y220C reactivator being evaluated in clinical trials. Rezatapopt was specifically designed to tightly bind to a pocket created by the TP53 Y220C mutation. By stabilization of the p53 protein structure, rezatapopt restores p53 tumor suppressor functions. In mouse models with established human tumor xenografts harboring the TP53 Y220C mutation, rezatapopt demonstrated tumor inhibition and regression at well-tolerated doses. In Phase 1 clinical trials, rezatapopt demonstrated a favorable safety profile within the efficacious dose range and showed single-agent efficacy in heavily pretreated patients with various TP53 Y220C mutant solid tumors.

Phase I analysis from the PYNNACLE phase I/II study of PC14586 in the subgroup of patients with advanced ovarian cancer harboring a TP53 Y220C mutation

Phase I analysis from the PYNNACLE phase I/II study of PC14586 in the subgroup of patients with advanced ovarian cancer harboring a TP53 Y220C mutation

691TiP PYNNACLE phase II trial of rezatapopt (PC14586) in solid tumors with a TP53 Y220C mutation

691TiP PYNNACLE phase II trial of rezatapopt (PC14586) in solid tumors with a TP53 Y220C mutation

Design, Synthesis, and Evaluation of p53Y220C Acetylation Targeting Chimeras (AceTACs).

The well-known tumor suppressor p53 is mutated in approximately half of all cancers. The Y220C mutation is one of the major p53 hotspot mutations. Several small-molecule stabilizers of p53Y220C have been developed. We recently developed a new technology for inducing targeted protein acetylation, termed acetylation targeting chimera (AceTAC), and the first p53Y220C AceTAC that effectively acetylated p53Y220C at lysine 382. Here, we report structure-activity relationship (SAR) studies of p53Y220C AceTACs, which led to the discovery of a novel p53Y220C AceTAC, compound 11 (MS182). 11 effectively acetylated p53Y220C at lysine 382 in a time- and concentration-dependent manner via inducing the ternary complex formation between p300/CBP acetyltransferase and p53Y220C. 11 was more effective than the parent p53Y220C stabilizer in suppressing the proliferation and clonogenicity in cancer cells harboring the p53Y200C mutation and was bioavailable in mice. Overall, 11 is a potentially valuable chemical tool to investigate the role of p53Y220C acetylation in cancer.

Reconnaissance of Allostery via the Restoration of Native p53 DNA-Binding Domain Dynamics in Y220C Mutant p53 Tumor Suppressor Protein.

Allosteric regulation of protein dynamics infers a long-range deliberate propagation of information via micro- and macroscale interactions. The Y220C structural mutant is one of the most frequent cancerous p53 mutants. The mutation is distally located from the DNA-binding site of the p53 DNA-binding domain yet causes changes in DNA recognition. This system presents a unique opportunity to examine the allosteric control of mutated proteins under a drug design paradigm. We focus on the key case study of p53 Y220C mutation restoration by a series of new compounds suggested to have Y220C reactivation properties in comparison to our previous findings on the restorative potential of PK11000, a compound studied extensively for reactivation in vitro and in vivo. Previously, we implemented all-atom molecular dynamics (MD) simulations and our lab's techniques of MD-Sectors and MD-Markov state models on the wild type, the Y220C mutant, and Y220C with PK11000 to characterize the effector's restorative properties in terms of conformational dynamics and hydrogen bonding. In this study, we turn to probing the effects made by docking the battery of a new but less well-tested set of aminobenzothiazole derivative compounds reported by Baud et al., which show promise of Y220C rescue. We find that while complete and precise reconstitution of p53 WT molecular dynamics may not be observed as was the case with PK11000, dispersed local reconstitution of loop dynamics provides evidence of rescuing effects by aminobenzothiazole derivative N,2-dihydroxy-3,5-diiodo-4-(1H-pyrrol-1-yl)benzamide, Effector 22, like what we observed for PK11000. Generalizable insights into the mutation and allosteric reactivation of p53 by various effectors by reconstitution of WT dynamics observed in statistical conformational ensemble analysis and network inference are discussed, considering the development of allosteric drug design rooted in first principles.

Abstract 7275: GS-P-328, a brain-penetrant small molecule p53 Y220C reactivator for tumors harboring p53 Y220C mutation

Abstract As a tumor suppressor, p53 can prevent tumor development through multiple pathways. However, mutation of TP53 and the resultant inactivation of p53 allow evasion of tumor cell death and rapid tumor progression. The oncogenic Y220C mutation is the ninth most frequent p53 missense mutation in cancer. Y220C mutation creates a narrow, hydrophobic pocket on the surface of the p53 DBD that reduces its thermal stability. As a result, the Y220C mutation causes a rapidly unfold of the p53 protein under physiological conditions, abrogates p53 signaling and drives tumorigenesis. There are approximately 100,000 new Y220C mutation cancer cases per year worldwide with limited treatment choices. The initial clinical evaluation of PC14586 that can restore missense-mutant p53 protein has seen efficacy in patients harboring p53 Y220C mutation even though there is still room for improvement. Here we report a brain-penetrable, highly potent p53 Y220C reactivator which has great in vitro activity as well as induce tumor shrinkage in multiple tumor models at a much lower systematic exposure comparing to PC14586. In vitro, GS-P-328 could bind to p53 Y220C protein with a single nM potency and potently induce cell death in a wide range of p53 Y220C tumor cells while have no anti-proliferative effects with p53 WT cells. GS-P-328 is also active in patient-derived cell (PDC) models in various tumor background including SCLC, gastric, ovarian and colon. In vivo, GS-P-328, at approximately 1/3 of systematic exposure comparing to a leading clinical reactivator, causes tumor shrinkage in multiple CDX models. GS-P-328 could induce MDM2/p21 upregulation in cell systems. In vivo, GS-P-328 could induce 2 fold and more increase in MDM2/p21 protein levels in tumor tissues at approximately 1/3 of plasma concentrations comparing to PC14586. More importantly, in a NUGC3-luc intracranial model, GS-P-328 could strongly inhibit growth of cancer cells in the mouse brain. GS-P-328 has a favorable ADME profile in rodents, dogs and NHPs, as well as a much better safety profile. GS-P-328 is now under IND-enabling study and P1 study is planned in early 2025. Citation Format: Meng Liu, Kaijun Geng, Biao Lu, Yuanfeng Xia, Fanglong Yang. GS-P-328, a brain-penetrant small molecule p53 Y220C reactivator for tumors harboring p53 Y220C mutation [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 7275.

Abstract LB_A25: Updated Phase 1 results from the PYNNACLE Phase 1/2 study of PC14586, a selective p53 reactivator, in patients with advanced solid tumors harboring a TP53 Y220C mutation

Abstract Background: Mutations in the TP53 gene leading to p53 inactivation are the most common mutational event across human cancers. PC14586 is a first-in-class p53 reactivator that selectively binds to the mutated p53 Y220C protein and restores p53 wild-type activity. Preclinical data support potential for activity of PC14586 across TP53 Y220C-bearing tumors, with enhanced activity in KRAS wild-type (WT) models, consistent with the important parallel roles for p53 inactivation and oncogenic KRAS in tumorigenesis. Preliminary analysis of Phase 1 data from the Phase 1/2 PYNNACLE trial (NCT04585750) evaluating PC14586 in patients with advanced TP53 Y220C solid tumors showed that PC14586 was well tolerated with preliminary clinical activity across multiple tumor types in the efficacious dose range (1150 mg QD to 1500 mg BID). This updated Phase 1 analysis assesses PC14586 in patients treated across the efficacious dose range. Methods: Eligible patients (≥12 years) with locally advanced or metastatic solid tumors with a TP53 Y220C mutation received increasing oral doses of PC14586 to evaluate safety, PK, and preliminary efficacy via RECIST v1.1, and to determine the Recommended Phase 2 Dose (RP2D). Tumor molecular profiling was assessed for the impact of KRAS status on response. Results: As of 1 May 2023, 61 patients were treated in the efficacious dose range (safety population): median age 63 (range 32-84) years, 62% female, and median of 3 (range 1–9) prior lines of systemic therapy. The most frequent treatment-related adverse events (TRAEs) (≥10%) were nausea (42.3%), vomiting (35.2%), diarrhea (19.7%), increased creatinine (18.3%), fatigue (14.1%), and increased AST and ALT (12.7% each); 3% of patients discontinued PC14586 due to TRAEs. Most TRAEs were grade 1 or 2; the most common grade 3 TRAEs were anemia (4.2%) and increased ALT (2.8%). PC14586 administered with food led to improvement in nausea and vomiting. Dose proportional and linear PK was observed with a median half-life of 19 hours. Among patients with measurable disease, 36 patients had KRAS WT and 15 patients had KRAS mutated tumors. A total of 12 PRs (ORR 33.3%) were observed (9 confirmed and 3 unconfirmed - pending confirmation) in patients with KRAS WT disease across multiple tumor types including ovarian, breast, prostate, small-cell lung, and endometrial cancer. The median duration of confirmed response was 7 months. In patients with a KRAS mutation, there were no confirmed responses, although clinical activity was observed. At 2000 mg QD, the ORR was 46.2% in patients with TP53 Y220C and KRAS WT tumors (n=13). A RP2D of 2000 mg QD was selected based on overall safety, PK and preliminary efficacy. Conclusions: PC14586 was well-tolerated with a favorable safety profile. Efficacy was achieved in heavily pre-treated patients across multiple tumor types. The PYNNACLE registrational Phase 2 trial will assess PC14586 as monotherapy at the RP2D of 2000 mg QD in patients with TP53 Y220C and KRAS WT advanced solid tumors. Additional/updated data will be presented at the conference. Citation Format: Alison M Schram, Geoffrey I Shapiro, Melissa L Johnson, Anthony W. Tolcher, John A Thompson, Anthony B El-Khoueiry, Andrae L Vandross, Shivaani Kummar, Aparna R Parikh, Dale R Shepard, Ursula Garczarek, Kim LeDuke, Lisa Sheehan, Marc Fellous, Leila Alland, Ecaterina E Dumbrava. Updated Phase 1 results from the PYNNACLE Phase 1/2 study of PC14586, a selective p53 reactivator, in patients with advanced solid tumors harboring a TP53 Y220C mutation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A25.

TP53 Y220C Mutations in Patients with Myeloid Malignancies

Background: TP53 gene mutations lead to inactivation of the protein p53, causing genome instability, oncogenesis, and treatment resistance conferring an unfavorable prognosis. TP53 Y220C missense mutations in exon 6 (p.659A>G) are described as a frequent ‘hot spot’ mutation in solid tumors. A novel Y220C-targeted small molecule (PC14586) has shown preliminary efficacy and safety in patients with solid tumors (Dumbrava, E., ASCO 2022). No data has been reported about TP53 Y220C in hematologic malignancies to date. We set out to evaluate the clinicopathologic characteristics of TP53 Y220C in hematologic malignancies and particularly, myeloid neoplasms. Methods: We identified all patients with hematologic neoplasms with TP53 Y220C mutations at University of Texas MD Anderson Cancer Center and Memorial Sloan-Kettering Cancer Center, detected by standard next generation sequencing assays, and focused on patients with myeloid malignancies; summarizing the patient characteristics, disease features and outcomes for patients with this specific TP53 mutation. Overall survival (OS) was assessed by the Kaplan-Meier method from date of diagnosis to date of death from any cause or date of last follow-up. Leukemia-free survival (LFS) was calculated from time to diagnosis of TP53 Y220C mutated myelodysplastic syndrome (MDS) to date of transformation to acute myeloid leukemia (AML) or death. Survival analysis was conducted only for newly diagnosed patients with available follow-up data. Results: We identified 143 patients (pts) with hematologic malignancies harboring a TP53 Y220C mutation. MDS and AML were the most frequent diagnoses (MDS, n=66; AML, n=48). Median age was 69 years (range, 32-92) and 56% of patients were male (Table 1). We then analyzed in more detail patients with MNs, including CHIP/CCUS, MDS, AML and MDS/MPN. Among those, in 76 (64%) pts the TP53 Y220C mutation was detected at initial diagnosis and in 43 (36%) pts, TP53 Y220C was identified in the relapsed/refractory setting. Therapy-related myeloid neoplasms (MNs) accounted for 51 of 119 pts (41%). Complex karyotype was frequent (n=88/119, 74%) in the setting of TP53 Y220C. Median TP53 Y220C variant allele frequency (VAF) was 32% (range, 1%-96%). Eighty-two patients (69%) had additional co-mutations: 24 (20%) pts had additional (non-Y220C) TP53 mutations only; 34 (29%) pts had only non- TP53 co-mutations (most frequently DNMT3A and TET2) and 24 (20%) pts had additional TP53 mutations in the setting of other co-mutations. With a median follow-up of 9 months, median OS for patients with newly diagnosed TP53 Y220C MDS (n=39) and AML (n=23) was 13.1 months (95% CI, 7.6 to 18.7) and 8 months (95% CI, 5.6 to 13.4), respectively ( p=0.06) (Figure 2). For the TP53 Y220C MDS cohort, AML transformation occurred in 13 (33%) patients, after a median of 14.3 months. Median LFS for these patients was 12.2 months (95% CI, 7.9 to 16.5). Modest and non-statistically significant differences in median LFS for MDS patients who did and did not receive stem cell transplant (SCT) were identified; MDS+SCT (n=12) 14.5 months (95% CI, 4 to 25) vs MDS+no-SCT (n=27) 9.3 months (95% CI, 4.6 to 14), p=0.18. Interestingly, four patients diagnosed with TP53 Y220C mutated CHIP/CCUS with a median VAF of 2% were identified, and with a median follow-up of 34.6 months, none have developed dysplastic changes or progression to MDS or AML at the time of cut off. Additional outcomes data will be forthcoming in the analysis of this cohort of patients with TP53 Y220C mutated myeloid neoplasms. Conclusions: TP53 Y220C mutations are present in malignant blood disorders and are particularly more common in myelodysplastic syndromes and acute myeloid leukemia, with associated poor outcomes. Novel targeted therapies for this TP53 variant (Y220C) would be of interest for this patient population.

In This Issue

In This Issue| January 09 2023 In This Issue Author & Article Information Online Issn: 2159-8290 Print Issn: 2159-8274 ©2023 American Association for Cancer Research2023American Association for Cancer Research Cancer Discov (2023) 13 (1): 1–3. https://doi.org/10.1158/2159-8290.CD-13-1-ITI Related Content A commentary has been published: A Molecular Switch between Mammalian MLL Complexes Dictates Response to Menin–MLL Inhibition A commentary has been published: Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance A commentary has been published: Molecular Characterization of Acquired Resistance to KRASG12C–EGFR Inhibition in Colorectal Cancer View more A commentary has been published: A Small Molecule Reacts with the p53 Somatic Mutant Y220C to Rescue Wild-type Thermal Stability A commentary has been published: Human Papillomavirus 42 Drives Digital Papillary Adenocarcinoma and Elicits a Germ Cell–like Program Conserved in HPV-Positive Cancers A commentary has been published: Creating MHC-Restricted Neoantigens with Covalent Inhibitors That Can Be Targeted by Immune Therapy A commentary has been published: Osimertinib + Savolitinib to Overcome Acquired MET-Mediated Resistance in Epidermal Growth Factor Receptor–Mutated, MET-Amplified Non–Small Cell Lung Cancer: TATTON A commentary has been published: Cell Competition Shapes Metastatic Latency and Relapse A commentary has been published: Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety A commentary has been published: Identifying the Transcriptional Drivers of Metastasis Embedded within Localized Melanoma A commentary has been published: An Aged/Autoimmune B-cell Program Defines the Early Transformation of Extranodal Lymphomas View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record January 9 2023 Citation In This Issue. Cancer Discov 1 January 2023; 13 (1): 1–3. https://doi.org/10.1158/2159-8290.CD-13-1-ITI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search The combination of KRASG12C and EGFR inhibitors allows for the targeting of KRAS mutations in colorectal cancer, but acquired resistance limits the duration of response. Yaeger and colleagues characterized mechanisms of resistance to KRASG12C plus EGFR inhibitors and found that heterogenous resistance changes largely converge on reactivation of ERK signaling. Moreover, KRASG12C amplification is a recurrent resistance mechanism that is positively selected for with treatment. When treatment is stopped, the acquired KRASG12C amplification leads to oncogene-induced senescence due to supraphysiologic ERK signaling and high mTOR signaling, opening the potential for new therapeutic approaches targeting this senescence. See article, p. 41. The tumor suppressor/transcription factor p53 is the most frequently mutated and inactivated gene in cancer. The p53 Y220C mutation occurs in 1% of patients and results in a thermally unstable protein that unfolds at physiologic temperatures. In this study, Guiley and Shokat discovered compounds that covalently... You do not currently have access to this content.

A Descriptive Analysis of TP53 Y220C Mutations in Patients with Hematologic Malignancies

Background: Mutations in the key tumor suppressor gene TP53 lead to inactivation of the protein p53, which leads to genome instability, oncogenesis, treatment resistance and an overall unfavorable prognosis in various malignancies. The specific TP53 Y220C variant has been described as one of the most frequent "hot spot" mutations associated with solid cancers. Recently, a Phase 1 clinical trial studying the novel Y220C-targeted small molecule PC14586, has shown preliminary safety and efficacy in patients with heavily treated solid tumors. No data has been reported about TP53 Y220C in hematologic malignancies to date. The aim of the present analysis is to evaluate the clinicopathologic characteristics of TP53 Y220C in hematologic malignancies. Methods: We searched our molecular laboratory database for hematologic neoplasms with TP53 Y220C mutations and summarized the patient characteristics, disease features and outcomes for patients treated within the Department of Leukemia at MD Anderson. Overall survival (OS) was assessed by the Kaplan-Meier method calculated from the date of TP53 Y220C detection to the date of death from any cause or date of last follow-up. Results: We identified 126 patients with hematologic malignancies harboring a TP53 Y220C mutation. Nine patients with lymphoma or myeloma were treated outside our Department and were excluded from subsequent analysis. 117 patients had a diagnosis of acute or chronic leukemia or myelodysplastic syndrome (MDS) [Table 1]. Median age was 69 years (31-91) and 59% of patients were male. Acute myeloid leukemia (AML) and MDS accounted for 33% and 38%, respectively. Consistent with the increased incidence in TP53-mutated myeloid disorders, 28% of AML and 43% of MDS cases were therapy-related. Cytogenetics was abnormal in 85 patients (73%), with a complex karyotype occurring in 70 patients (60%). Among 73 patients with detailed VAF data, the TP53 Y220C mutation had a median VAF of 35% (1-96%). Seventy-eight patients (67%) had additional co-mutations; 12 patients (10%) had only additional TP53 mutations and the remaining 66 patients (57%) had additional variants involving genes other than TP53 (56 patients with known somatic mutations and 10 patients with variants of uncertain significance -VUS). With a median follow-up of 49 months, the median OS of patients with TP53 Y220C mutations was 10 months, recognizing that the treatments received were variable based on diagnosis, age, and comorbidities among other variables. Median OS was 6.7, 9.3 and 60 months for AML, MDS and CLL patients, respectively. Additional outcomes data will be forthcoming. Conclusions: TP53 Y220C mutation is present in hematologic diseases, primarily in myeloid neoplasms (MDS and AML) and with higher frequency in therapy-related disease. Targeted therapies for this specific TP53 mutation would be of interest in this difficult to treat patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

76P Modulation of mutant p53 activity using indazole derivatives

Oncogenic mutation p53 Y220C is the ninth most common mutation of p53 protein. The Y220C mutation creates an expanded surface pocket in the DNA-binding domain, rapidly unfolds and denatures under physiological conditions, which effectively cancels p53 signaling and leads to the development of a tumor. In this work the novel indazole derived compounds were tested. It was found that these molecules bind to the mutated p53 protein and stabilize its structure in vitro.

First-in-human study of PC14586, a small molecule structural corrector of Y220C mutant p53, in patients with advanced solid tumors harboring a TP53 Y220C mutation.

3003 Background: The p53 tumor suppressor protein is a transcription factor that acts to maintain genome stability in response to cellular stress. Spontaneous mutation of the TP53 gene leading to inactivation of the p53 protein is the most common mutational event across all human cancers. PC14586 is a novel, small molecule structural corrector that binds selectively to p53 Y220C mutant protein and restores the p53 wildtype conformation and transcriptional activity, resulting in potent preclinical antitumor activity. This Phase 1 multicenter dose escalation study assesses PC14586 safety, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary efficacy in patients (pts) with advanced solid tumors that harbor the TP53 Y220C mutation. Methods: Eligible adult pts with locally advanced or metastastic TP53 Y220C mutant solid tumors received increasing doses of oral PC14586 using the modified Toxicity Probability Interval design to estimate toxicity and to determine maximum tolerated dose and recommended phase 2 dose. Plasma PK was characterized using standard methods. Preliminary efficacy was assessed by RECIST v1.1. Reporting of interim results was approved by the study’s Safety Review Committee. Results: As of 08 Feb 2022, 29 pts (62% female, median age 62 years) with a variety of TP53 Y220C mutant solid tumor types (median number of prior lines of therapy 3; range 1 to 8) were treated in 7 dose cohorts of PC14586: 150 mg QD (3 pts), 300 mg QD (3 pts), 600 mg QD (4 pts), 1150 mg QD (5 pts), 2000 mg QD (7 pts), 2500 mg QD (4 pts) and 1500 mg BID (3 pts). PC14586 was generally well-tolerated; treatment-related AEs were observed in 79% of pts that were all Grade 1/2 in severity except 2 Grade 3 AEs (alanine aminotransferase increased and neutrophil count decreased). The most common AEs (≥15% of pts) were nausea (34%), vomiting (24%), fatigue (21%), and aspartate aminotransferase increased (17%). There were no dose limiting toxicities and enrollment continues. PK analysis showed dose proportional increases in Cmax and AUC. Amongst 21 efficacy evaluable pts, PRs were observed in 5 pts: 1 small cell lung and 1 breast with confirmed PR (cPR), both ongoing; 1 colorectal with unconfirmed PR (uPR), and 2 prostate with uPR and ongoing. In the 3 highest dose cohorts (total daily dose 2000 to 3000 mg), there were 3 PRs (2 uPR, 1cPR) and 7 SD out of 10 efficacy evaluable pts (all ongoing). Observations of decreasing p53 Y220C circulating tumor DNA and decreasing numbers of circulating tumor cells in pts further support on-target anti-tumor activity of PC14586. Conclusions: Enrollment to a Phase 1 study is feasible in a TP53 mutation selective population. PC14586 is safe and tolerated up to 3000 mg daily. Preliminary efficacy was achieved in heavily pretreated pts. Additional safety, PK, PD and efficacy data will be reported at the annual meeting. Clinical trial information: NCT04585750.

Insights into Rational Design of a New Class of Allosteric Effectors with Molecular Dynamics Markov State Models and Network Theory.

The development of drugs to restore protein function has been a major advance facilitated by molecular medicine. Allosteric regulation, a phenomenon widely observed in nature, in which a molecule binds to control a distance active site, holds great promise for regulating proteins, yet how to rationally design such a molecule remains a mystery. Over the past few years, we and others have developed several techniques based on molecular dynamics (MD) simulations: MD-Markov state models to capture global conformational substates, and network theory approach utilizing the interaction energy within the protein to confer local allosteric control. We focus on the key case study of the p53 Y220C mutation restoration by PK11000, a compound experimentally shown to reactivate p53 native function in Y220C mutant present tumors. We gain insights into the mutation and allosteric reactivation of the protein, which we anticipate will be applicable to de novo design to engineer new compounds not only for this mutation, but in other macromolecular systems as well.

Abstract LB006: PC14586: The first orally bioavailable small molecule reactivator of Y220C mutant p53 in clinical development

Abstract Mutations in the TP53 gene are the most frequent somatic alterations in human cancer; approximately 50% of all human cancers possess mutations in TP53. Missense mutations in TP53 which result in a non-functional protein are the most frequently identified, these occur across the gene, however most hotspot mutations are localized within the DNA binding domain of the protein. Y220C is one such hotspot mutation, prevalent in ~1.8% of TP53 mutant tumors. PC14586 was structurally designed to bind tightly to a crevice within the mutant protein (KD~2.5 nM). It is the first orally bioavailable small molecule and selective reactivator of Y220C mutant p53 protein in clinical development. In Y220C mutant human cell lines, PC14586 was shown to stabilize the Y220C mutant in the wild type conformation, resulting in reactivation of p53 transcriptional activity and subsequent expression of its target proteins (e.g. p21, MDM2, Bax, PUMA). The reactivation of p53 function is highly selective to Y220C mutant cells and results in arrest of the cell cycle in vitro (IC50 ~0.230-1.8 μM). PC14586 has favorable pharmaceutical properties in pre-clinical species. In nude mice bearing Y220C mutant NUGC3 gastric cancer xenograft tumors, oral administration of PC14586 results in a dose responsive anti-tumor effect, with a target efficacious dose of 100 mg/kg daily resulting in approximately 80% tumor regression after 3 weeks. This anti-tumor effect was driven by a dose responsive pharmacodynamic modulation of the mutant p53 target. In human xenografts, PC14586 was shown to convert Y220C mutant to the wildtype conformation, resulting in activation of p53 transcription. This was demonstrated by the measurement of a p53 mRNA transcriptional signature and at the protein level by increases in p21 and MDM2 in the tumor and Macrophage Inhibitory Cytokine-1 (MIC-1) in both the tumor and plasma. In a C57Bl/6J syngeneic xenograft model bearing the Y220C mutation, administration of PC14586 at 100 mg/kg orally resulted in complete tumor cure in 80% of mice. PC14586 is well tolerated by pre-clinical species and possesses a favorable development profile. The safety and efficacy of PC14586 is currently being evaluated in a seamless Phase I/II clinical study that is a biomarker driven, solid tumor agnostic trial with patients whose tumor bears the Y220C TP53 mutation (NCT study identifier NCT04585750). Citation Format: Melissa Dumble, Lizhong Xu, Romyr Dominique, Binbin Liu, Hong Yang, Mary-Kate McBrayer, Dafydd Thomas, Bruce Fahr, Hongju Li, Kuo-Sen Huang, Kimberly Robell, Chris Mulligan, Brandon Russo, Anna Puzio-Kuter, Thomas Davis, Binh Vu. PC14586: The first orally bioavailable small molecule reactivator of Y220C mutant p53 in clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB006.

Impact of tumor heterogeneity and microenvironment in identifying neoantigens in a patient with ovarian cancer.

Identification of neoepitopes as tumor-specific targets remains challenging, especially for cancers with low mutational burden, such as ovarian cancer. To identify mutated human leukocyte antigen (HLA) ligands as potential targets for immunotherapy in ovarian cancer, we combined mass spectrometry analysis of the major histocompatibility complex (MHC) class I peptidomes of ovarian cancer cells with parallel sequencing of whole exome and RNA in a patient with high-grade serous ovarian cancer. Four of six predicted mutated epitopes capable of binding to HLA-A*02:01 induced peptide-specific T cell responses in blood from healthy donors. In contrast, all six peptides failed to induce autologous peptide-specific response by T cells in peripheral blood or tumor-infiltrating lymphocytes from ascites of the patient. Surprisingly, T cell responses against a low-affinity p53-mutant Y220C epitope were consistently detected in the patient with either unprimed or in vitro peptide-stimulated T cells even though the patient's primary tumor did not bear this mutation. Our results demonstrated that tumor heterogeneity and distinct immune microenvironments within a patient should be taken into consideration for identification of immunogenic neoantigens. T cell responses to a driver gene-derived p53 Y220C mutation in ovarian cancer warrant further study.

A structure-guided molecular chaperone approach for restoring the transcriptional activity of the p53 cancer mutant Y220C.

Aim:The p53 cancer mutation Y220C creates a conformationally unstable protein with a unique elongated surface crevice that can be targeted by molecular chaperones. We report the structure-guided optimization of the carbazole-based stabilizer PK083.Materials & methods:Biophysical, cellular and x-ray crystallographic techniques have been employed to elucidate the mode of action of the carbazole scaffolds.Results:Targeting an unoccupied subsite of the surface crevice with heterocycle-substituted PK083 analogs resulted in a 70-fold affinity increase to single-digit micromolar levels, increased thermal stability and decreased rate of aggregation of the mutant protein. PK9318, one of the most potent binders, restored p53 signaling in the liver cancer cell line HUH-7 with homozygous Y220C mutation.Conclusion:The p53-Y220C mutant is an excellent paradigm for the development of mutant p53 rescue drugs via protein stabilization. Similar rescue strategies may be applicable to other cavity-creating p53 cancer mutations.

Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer.

Prion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimer's, Parkinson's, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others. Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases. Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53-MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP) and chemical denaturants have helped to clarify excited conformers of p53 that are prone to aggregation. Molecular dynamics (MD) and phasor analysis of single Trp fluorescence signals point toward the presence of preamyloidogenic conformations of p53, which are not observed for p63 or p73. Exploring the features of competent preamyloidogenic states of wt and different p53 mutants may provide a framework for designing personalized drugs for the restoration of p53 function. Protection of backbone hydrogen bonds (BHBs) has been shown to be an important factor for the stability of amyloidogenic proteins and was employed to identify and stabilize the structural defect resulting from the p53 Y220C mutation. Using MD simulations, we compared BHB protection factors between p53 family members to determine the donor-acceptor pairs in p53 that exhibit lower protection. The identification of structurally vulnerable sites in p53 should provide new insights into rational designs that can rapidly be screened using our experimental methodology. Through continued and combined efforts, the outlook is positive for the development of strategies for regulating p53 amyloid transformation.

An in silico algorithm for identifying stabilizing pockets in proteins: test case, the Y220C mutant of the p53 tumor suppressor protein.

The p53 tumor suppressor protein performs a critical role in stimulating apoptosis and cell cycle arrest in response to oncogenic stress. The function of p53 can be compromised by mutation, leading to increased risk of cancer; approximately 50% of cancers are associated with mutations in the p53 gene, the majority of which are in the core DNA-binding domain. The Y220C mutation of p53, for example, destabilizes the core domain by 4 kcal/mol, leading to rapid denaturation and aggregation. The associated loss of tumor suppressor functionality is associated with approximately 75 000 new cancer cases every year. Destabilized p53 mutants can be 'rescued' and their function restored; binding of a small molecule into a pocket on the surface of mutant p53 can stabilize its wild-type structure and restore its function. Here, we describe an in silico algorithm for identifying potential rescue pockets, including the algorithm's integration with the Dynameomics molecular dynamics data warehouse and the DIVE visual analytics engine. We discuss the results of the application of the method to the Y220C p53 mutant, entailing finding a putative rescue pocket through MD simulations followed by an in silico search for stabilizing ligands that dock into the putative rescue pocket. The top three compounds from this search were tested experimentally and one of them bound in the pocket, as shown by nuclear magnetic resonance, and weakly stabilized the mutant.

FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53.

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP1 expression in HCC tumors with the CHC background. Oncomine data analysis of normal versus HCC tumors with the CHC background indicated a 4-fold increase in FBP1 expression with a concomitant 2.5-fold decrease in the expression of p53. We found that FBP1 promotes HCV replication by inhibiting p53 and regulating BCCIP and TCTP, which are positive and negative regulators of p53, respectively. The severe inhibition of HCV replication in FBP1-knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that the activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP1 but significantly activated upon knockdown of FBP1. Transient expression of FBP1 in FBP1 knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), we found no significant difference in in vitro target DNA binding affinity of recombinant wild-type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP1, the DNA binding ability of p53 is strongly inhibited. We confirmed that FBP1 downregulates BCCIP, p21, and p53 and upregulates TCTP under radiation-induced stress. Since FBP1 is overexpressed in most HCC tumors with an HCV background, it may have a role in promoting persistent virus infection and tumorigenesis. It is our novel finding that FUSE binding protein 1 (FBP1) strongly inhibits the function of tumor suppressor p53 and is an essential host cell factor required for HCV replication. Oncomine data analysis of a large number of samples has revealed that overexpression of FBP1 in most HCC tumors with chronic hepatitis C is significantly linked with the decreased expression level of p53. The most significant finding is that FBP1 not only physically interacts with p53 and interferes with its binding to the target DNA but also functions as a negative regulator of p53 under cellular stress. FBP1 is barely detectable in normal differentiated cells; its overexpression in HCC tumors with the CHC background suggests that FBP1 has an important role in promoting HCV infection and HCC tumors by suppressing p53.