Hotspot Mutants Research Articles

The PPP2R1A cancer hotspot mutant p.R183W increases clofarabine resistance in uterine serous carcinoma cells by a gain-of-function mechanism.

Uterine serous carcinoma (USC) is generally associated with poor prognosis due to a high recurrence rate and frequent treatment resistance; hence, there is a need for improved therapeutic strategies. Molecular analysis of USC identified several molecular markers, useful to improve current treatments or identify new druggable targets. PPP2R1A, encoding the Aα subunit of the tumor suppressive Ser/Thr phosphatase PP2A, is mutated in up to 40% of USCs. Here, we investigated the effect of the p.R183W PPP2R1A hotspot variant on treatment response to the nucleoside analogue clofarabine. USC cells stably expressing p.R183W Aα showed increased resistance to clofarabine treatment in vitro and, corroborated by decreased clofarabine-induced apoptosis, G1 phase arrest, DNA-damage (γH2AX) and activation of ATM and Chk1/2 kinases. Phenotypic rescue by pharmacologic PP2A inhibition or dicer-substrate siRNA (dsiRNA)-mediated B56δ subunit knockdown supported a gain-of-function mechanism of Aα p.R183W, promoting dephosphorylation and inactivation of deoxycytidine kinase (dCK), the cellular enzyme responsible for the conversion of clofarabine into its bioactive form. Therapeutic assessment of related nucleoside analogues (gemcitabine, cladribine) revealed similar effects, but in a cell line-dependent manner. Expression of two other PPP2R1A USC mutants (p.P179R or p.S256F) did not affect clofarabine response in our cell models, arguing for mutant-specific effects on treatment outcome as well. While our results call for PPP2R1A mutant and context-dependent effects upon clofarabine/nucleoside analogue monotherapy, combining clofarabine with a pharmacologic PP2A inhibitor proved synergistically in all tested conditions, highlighting a new generally applicable strategy to improve treatment outcome in USC.

RNA expression-based risk stratification of stage III cutaneous melanoma.

e21551 Background: Prognostication of locally advanced cutaneous melanoma (CM) relies on accurate assessment of primary tumor invasion depth and extent of lymph node involvement. However, precision is often limited by detection limits of positron emission computed tomography and sentinel lymph node biopsy, and the estimated disease-specific survival (DSS) can vary widely. In this study, we explored tumor gene expression-based risk stratification strategies and found that CDH1 (E-cadherin) and BRAF transcript level profiles can predict DSS in stage III CM. Methods: The Cancer Genome Atlas Skin Cutaneous Melanoma (TCGA-SKCM) project’s normalized RNA-Seq transcriptome profile (n = 288) was analyzed with MATLAB software. TCGA-SKCM adopted AJCC 7th edition staging. "High” or “low” level of each gene was determined by the median FPKM of the cohort used as cutoff. P-values were obtained from Cox proportional hazards regression model. Results: In stage III (n = 96), 3-year DSS for high- and low-CDH1 groups were 45% and 75%, respectively (p = 1.5x10-5). High-BRAF group was weakly associated with superior 3-year DSS compared to low-BRAF group (75% vs. 55%; p = 0.06), although high-BRAF group was composed of more BRAF hotspot mutants compared to low-BRAF group (56% vs. 30%). The 3-year DSS for high-CDH1/low-BRAF and low-CDH1/high-BRAF groups were 28% and 84%, respectively (p = 4.4x10-5). Composition of stages [IIIA, IIIB, IIIC, IIINOS] in high-CDH1/low-BRAF and low-CDH1/high-BRAF groups were [5%, 18%, 45%, 32%] and [23%, 13%, 37%, 27%], respectively. For high-CDH1/high-BRAF group, 3-year DSS was 54% with stage composition [6%, 33%, 39%, 22%]. DSS was not associated with CDH1/BRAF levels in stage I and II. Stage IV was not analyzed due to the small sample size. Conclusions: We demonstrate CDH1/BRAF transcript level-based prognostication of CM cases with regional metastasis (metastatic node, in-transit or satellite metastasis) defined as stage III. Our findings are contrary to the notion that CDH1 is a tumor/invasion suppressor gene, although we note the emerging evidence that CDH1 expression boosts establishment of cancer at a secondary site once they escape from the primary site1). The association between high BRAF level and superior survival may also seem counterintuitive given the well-accepted oncogenicity of activating BRAF mutations in variety of malignancies. However, we speculate that wild-type and oncogenic BRAF, which RNA-Seq cannot easily differentiate, may play independent roles in tumorigenesis similar to what has been previously reported in wild-type and oncogenic RAS2).

Abstract LT10: Recurrent FBXW7 Mutations Bypass Wnt/β-catenin Addiction in Cancer

Abstract Inactivating mutations in RNF43 or gene fusions involving RSPO2/3 are potent activators of ligand-dependent Wnt signaling and drive multiple human cancers. Pharmacologically targeting the biogenesis of Wnt ligands, e.g., with PORCN inhibitors, is being investigated in clinical trials for treating RNF43-mutant/RSPO-fusion cancers. But, the therapeutic response is heterogeneous. Here we show that the tumor suppressor FBXW7 is frequently mutated in RNF43-mutant/RSPO-fusion tumors. Multiple RNF43-mutant/RSPO-fusion cancers harboring FBXW7 mutations show resistance to Wnt pathway blockade, while silencing FBXW7 by truncation or expressing a hotspot mutant in FBXW7-wildtype tumors leads to intrinsic drug resistance. Mechanistically, inactivation of FBXW7, the substrate recognition component of a SCF E3 ubiquitin ligase complex, stabilizes multiple oncoproteins especially Cyclin E and MYC, and antagonizes the cytostatic effect of Wnt inhibitors in these cancers. Moreover, although FBXW7 mutations do not mitigate β-catenin degradation upon Wnt inhibition, FBXW7 mutants instead lose dependence on β-catenin signaling, accompanied by dedifferentiation and loss of lineage specificity. These FBXW7-mutant Wnt/β-catenin-independent tumors are susceptible to multi-CDK inhibition by dinaciclib that targets both cell cycle and transcription. In summary, this study identifies FBXW7 mutations as a mechanism of Wnt independence and drug resistance in RNF43-mutant/RSPO-fusion cancers and reveals the potential of treating these cancers with multi-CDK inhibitors. Citation Format: Zheng Zhong, David Virshup. Recurrent FBXW7 Mutations Bypass Wnt/β-catenin Addiction in Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT10.

Abstract 4439: Decoding the metabolic symphony TP53 mutation-driven rewiring of cellular metabolism in non-small cell lung carcinoma

Abstract TP53 is a critical regulator of major metabolic pathways. Metabolic reprogramming is one of the "hallmarks of cancer" and drives tumorigenesis. Frequent p53mutations not only eradicate tumour suppressor capacities but also confer various activities that impact the alteration of metabolic pathways now regarded as central for tumour development and progression. We selected three hotspot mutants (R175H, R273H and R249S) and wtp53 to evaluate the effects in the human non-small cell lung carcinoma cell line (NCI-H1299). Differential expressions of p53 mutants were observed in normal and glucose/glutamine starvation. However, WTp53 expression was not found to be affected. Under both the glucose and glutamine starvation, inhibition of the cell growth and migratory potential of mutant cells was observed which was more prominent in glutamine starvation. Basal respiration and ATP production in p53-/- R273H and R249S significantly increased under glucose starvation. However, Non-mitochondrial oxygen consumption and maximal respiration were found to be decreased in all the cell lines. Importantly, the cells harbouring mutation R175H increase the glycolysis and TCA cycle. The metabolic intermediates of the urea cycle were found to be increased by nearly fourfold in R273H cells. Indicating increased utilization of the urea cycle instead of the TCA cycle, which has been known to be fueled by glutamine for anaplerosis. This study demonstrates that mutp53 influences several metabolic processes and has an influence on cancer cell aggression under starvation of main carbon sources (Glucose and Glutamine). In starved conditions, cells with p53 mutations enhance mitochondrial activity and use alternative pathways in non-small cell lung cancer cells. This might provide a foundation for the development of more effective targeted therapeutics/pharmacological approaches toward variants of mutant p53. Citation Format: Jiyauddin Khan, Asad Ur Rehman, Ankit Mathur, Naveen Kumar Bhatrajuc, Anannya Tulid, Divya Rajput, Mohammad Askandar Iqbal, Daman Saluja. Decoding the metabolic symphony TP53 mutation-driven rewiring of cellular metabolism in non-small cell lung carcinoma [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 4439.

Jun Dimerization Protein 2 (JDP2) Increases p53 Transactivation by Decreasing MDM2.

The AP-1 protein complex primarily consists of several proteins from the c-Fos, c-Jun, activating transcription factor (ATF), and Jun dimerization protein (JDP) families. JDP2 has been shown to interact with the cAMP response element (CRE) site present in many cis-elements of downstream target genes. JDP2 has also demonstrates important roles in cell-cycle regulation, cancer development and progression, inhibition of adipocyte differentiation, and the regulation of antibacterial immunity and bone homeostasis. JDP2 and ATF3 exhibit significant similarity in their C-terminal domains, sharing 60-65% identities. Previous studies have demonstrated that ATF3 is able to influence both the transcriptional activity and p53 stability via a p53-ATF3 interaction. While some studies have shown that JDP2 suppresses p53 transcriptional activity and in turn, p53 represses JDP2 promoter activity, the direct interaction between JDP2 and p53 and the regulatory role of JDP2 in p53 transactivation have not been explored. In the current study, we provide evidence, for the first time, that JDP2 interacts with p53 and regulates p53 transactivation. First, we demonstrated that JDP2 binds to p53 and the C-terminal domain of JDP2 is crucial for the interaction. Second, in p53-null H1299 cells, JDP2 shows a robust increase of p53 transactivation in the presence of p53 using p53 (14X)RE-Luc. Furthermore, JDP2 and ATF3 together additively enhance p53 transactivation in the presence of p53. While JDP2 can increase p53 transactivation in the presence of WT p53, JDP2 fails to enhance transactivation of hotspot mutant p53. Moreover, in CHX chase experiments, we showed that JDP2 slightly enhances p53 stability. Finally, our findings indicate that JDP2 has the ability to reverse MDM2-induced p53 repression, likely due to decreased levels of MDM2 by JDP2. In summary, our results provide evidence that JDP2 directly interacts with p53 and decreases MDM2 levels to enhance p53 transactivation, suggesting that JDP2 is a novel regulator of p53 and MDM2.

Abstract C090: Elucidation of misp53-driven subtype specification and functions in pancreatic cancer

Abstract This study examines how mutations in the TP53 gene, specifically gain-of-function (GOF) mutations, impact the subtyping and stratification of pancreatic ductal adenocarcinoma (PDAC). In addition, it investigates the underlying mechanisms as well as the potential therapeutic implications.TP53 mutations/deletions are present in 60-75 % of cases in PDAC. The most common mutations are three specific hotspot missense mutations (misp53) found at codons R273H, R248W, and R175H. Several studies indicate that these misp53 mutants, despite losing their tumor suppressive function, acquire new oncogenic effects promoting disease aggressiveness and resistance to conventional therapies. Recent findings show that misp53 mutants are associated with a poorly differentiated metastatic subtype in PDAC. However, it is unclear how distinct misp53 mutants, such as misp53R273H, affect PDAC progression and therapy resistance and whether different mutants exert distinct functions. In this study, we explore misp53R273H- and misp53R248W-driven subtype-specificity and functions in PDAC aggressiveness by exploiting transcriptomics, genomic binding and regulation, controlled functional pathways and potential therapeutic targets. By combining cell line-based RNA- and ChIP-seq data we report that misp53R273H associates with and fuels basal-like (BL) neoplastic features by a potential regulation of cell cycle progression. Preclinical mouse models underpinned these findings by connecting misp53R273H tumors to metastatic spread. Generation of constitutive knockdown cell lines and corresponding overexpression assays could support these findings by bridging a loss of misp53R273H to the more favorable classical subtype properties. Of note, co-immunoprecipitation coupled to mass spectrometry analysis revealed misp53-specific and common hotspot mutant interaction partners: for instance, proteins of the group of chromatin remodelers or the ubiquitination pathway that could serve as a mechanistic foundation for misp53-specific functional outcomes. Further, implementation of a large-scale drug screen, flow cytometry and protein expression analyses revealed insights in the potential misp53-specific regulated pathways and therapeutic vulnerabilities, which strongly correlate to cell cycle progression and thus to the previous results. Integrating these findings with preclinical mouse studies, patient-derived xenograft cell lines and publicly available patient data sets strengthens the reported results and highlights their translational relevance. In conclusion, this study clearly demonstrates that hotspot misp53 mutants play a critical role in influencing PDAC subtype-specification and disease aggressiveness. In particular, misp53R273H was associated with BL aggressiveness and cell cycle progression. The ultimate aim of the study is to identify misp53 mutant-specific patient stratification to broaden individualized therapy options. Citation Format: Laura Urbach, Lena Wieland, Frederike Penz, Lukas Klein, Jennifer Appelhans, Christof Lenz, Carolin Schneider, Schneider Günter, Ramona Schulz-Heddergott, Volker Ellenrieder, Elisabeth Heßmann, Shiv K. Singh. Elucidation of misp53-driven subtype specification and functions in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C090.

Abstract B102: Development of covalent inhibitors targeting Ras Q61 hotspot mutants

Abstract Mutations in the proto-oncogenes RAS are a key driver of various cancers, including pancreatic, colorectal, non–small cell lung cancer (NSCLC), melanoma, and thyroid cancer. Despite being considered undruggable for many years, recent efforts have led to the FDA approval of two drugs, Sotorasib and Adagrasib. They covalently target the K-Ras mutant isoform K-Ras(G12C), thereby inhibiting its oncogenic effect. However, the use of these drugs is inherently limited to cancers driven by the K-Ras(G12C) mutant, which is very common only in NSCLC. Although other covalent inhibitors targeting the K-Ras mutants K-Ras(G12S) and K-Ras(G12R) have been developed, it has so far not been possible to target the other frequently mutated amino acid Q61. Here we present our approach for the development of novel covalent inhibitors specifically targeting Q61 mutations in Ras proteins, which are prevalent in melanoma, multiple myeloma, and thyroid cancer. We demonstrate how covalent inhibitors specifically react with the mutant Q61C in both K and H-Ras proteins as proof-of-concept and show our efforts to extend this to the other Q61 mutant isoforms. We base our strategy on the modification of well-established switch-II pocket K-Ras inhibitors and explore multiple novel scaffolds specifically designed to target mutations of Q61. In addition to its occurrence in various cancers, Ras(Q61X) is also a common resistance mutation in patients treated with MAPK inhibitors. This work could further increase the number of patients potentially benefiting from Ras(Q61X) targeting drugs. Citation Format: Julius Pampel, Johannes Morstein, Qinheng Zheng, David M. Peacock, Kevan M. Shokat. Development of covalent inhibitors targeting Ras Q61 hotspot mutants [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B102.

CSIG-08. GAIN-OF-FUNCTION MUTANT P53 REGULATES LONG-NONCODING RNAS TO CONTROL HIF1Α TRANSCRIPTION IN GLIOBLASTOMA

Abstract P53 is mutated in a large subset of glioblastoma (GBM). Most hot-spot p53 mutants are gain-of-function (with only partially understood mechanisms). To investigate the role of gain-of-function mutant p53 (GOF-Mut-p53), we performed ChIP-seq analysis of GOF-Mut-p53 R273H, the most common p53 mutation in GBM. We identified and verified numerous GOF-Mut-p53 targets that differed from those of wildtype p53. Among the 2,830 targets of GOF-Mut-p53 were 535 long non-coding RNAs (lncRNAs), the majority of which we also found enriched in Mut-p53 human tumors and deregulated in the TCGA database. LncRNAs play important regulatory roles in cancer. One of the most highly enriched lncRNAs was LINC00643. We first performed ChIP confirmation of GOF-Mut-p53 binding to the promoter of LINC00643. We then showed that GOF-Mut-p53 inhibits LINC00643 expression. Furthermore, we found that LINC00643 is deregulated in human GBM and LGG and correlated with patient survival. Overexpression of LINC00643 led to a significant inhibition of GBM cell proliferation, migration, invasion and in vivo xenograft growth. Co-expression of human LINC00643 or its mouse homologue with PDGF and Cre in RCAS transgenic mice (N/tv-a; Ink4a-arf-/-; Pten-fl/fl) reduced tumor growth and improved animal survival. These data show that GOF-Mut-p53 exerts oncogenic effects in GBM by downregulating LINC00643. To elucidate the mechanisms of action of LINC00643, we performed Chromatin Isolation by RNA purification high-throughput sequencing (CHIRP-seq) to identify its binding targets. We found that LINC00643 binds to the HIF1α enhancer region. Overexpression of LINC00643 and knockdown of GOF-Mut-p53 in GBM cells under hypoxia growth conditions reduced HIF1α mRNA and protein expression. LINC00643 also regulated the expression of numerous hypoxia-regulating genes downstream of HIF1α. Our study shows for the first time that GOF-Mut-p53 transcriptionally regulates a subset of lncRNAs and that GOF-Mut-p53-regulated LINC00643 regulates hypoxia in GBM.

Elucidating the Mechanisms of R248Q Mutation-Enhanced p53 Aggregation and Its Inhibition by Resveratrol.

Aggregation of p53 mutants can result in loss-of-function, gain-of-function, and dominant-negative effects that contribute to tumor growth. Revealing the mechanisms underlying mutation-enhanced p53 aggregation and dissecting how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations are fundamentally important for the development of novel therapeutics for p53 aggregation-associated cancers. A recent experimental study shows that resveratrol (RSV) has an inhibitory effect on the aggregation of hot-spot R248Q mutant of the p53 core domain (p53C), while pterostilbene (PT) exhibits a relatively poor inhibitory efficacy. However, the conformational properties of the R248Q mutant leading to its enhanced aggregation propensity and the inhibitory mechanism of RSV against p53C aggregation are not well understood. Herein, we performed extensive all-atom molecular dynamics simulations on R248Q p53C in the absence and presence of RSV/PT, as well as wild-type (WT) p53C. Our simulations reveal that loop L3, where the mutation resides, remains compact in WT p53C, while it becomes extended in the R248Q mutant. The extension of loop L3 weakens the interactions between loop L3 and two crucial aggregation-prone regions (APRs) of p53C, leading to impaired interactions within the APRs and their structural destabilization as well as p53C. The destabilized APRs in the R248Q mutant are more exposed than in WT p53C, which is conducive to p53C aggregation. RSV has a higher preference to bind to R248Q p53C than PT. This binding not only stabilizes loop L3 of R248Q mutant to its WT-like conformation, preventing L3-extension-caused APRs' destabilization but also reduces APRs' solvent exposure, thereby inhibiting R248Q p53C aggregation. However, PT exhibits a lower hydrogen-bonding capability and a higher self-association propensity, which would lead to a reduced p53C binding and a weakened inhibitory effect on R248Q mutant aggregation. Our study provides mechanistic insights into the R248Q mutation-enhanced aggregation propensity and RSV's potent inhibition against R248Q p53C aggregation.

Abstract B014: Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS

Abstract Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most frequently mutated oncogene detected in 30% of all human cancers. Drugs that can impede the function of oncogenic KRAS offer exceptional therapeutic value. However, direct targeting KRAS is challenging due to its protein structure that lacks deep binding pockets for small-molecule inhibitors. Despite the recent success in targeting the G12C allele, targeted therapy for another hotspot mutant – G12V mutant KRAS has not been described. Here, we utilize CRISPR-Cas9 based genome-wide knockout screens and leverage a high-throughput microfluidic cell sorting platform to identify genes that regulate mutant KRAS harboring G12V mutation. The screens were conducted in two colorectal cancer cell lines HT29 and SW480, expressing wild-type KRAS and homozygous G12V KRAS, respectively. We have identified a list of genes whose loss-of-function reduces wild-type or mutant G12V KRAS protein expression. Our top gene identified from the mutant KRAS screen - denoted as selective-mutant KRAS modulator (SMKM) has shown promising results in selectively decreasing G12V KRAS protein expression. Through multiple assays including live cell imaging, western blot, flow cytometry, and proteomic analysis in SMKM knockout cells and SMKM inhibitor-treated cells. We found highly selective reduction in G12V KRAS protein expression. More importantly, genetically or pharmacologically inhibiting SMKM has demonstrated anti-tumor activity. We conducted cell viability assays including CellTiter-glo, MTT, and clonogenic assay. We treated a panel of G12V KRAS mutant and wild-type KRAS cell lines with SMKM inhibitor and found significantly reduced cell viability in G12V KRAS mutant cell lines across colorectal and lung cancer. To further demonstrate the downstream impact on mutant KRAS signaling, secondary assays including western blot and Lumit immunoassay were conducted. We observe a reduction in the aberrant phospho-ERK and phospho-AKT signaling in SMKM inhibitor treated cells. Our in vivo efficacy trial using heterozygous G12V KRAS lung cancer NCI-H441 xenograft model has shown SMKM inhibition significantly reduces tumor growth and increases the survival rate. Together, these data indicate the feasibility of selectively targeting G12V KRAS with SMKM inhibition. Lastly, our results provide a first-in-class small molecule oral inhibitor based selective protein clearance as an effective anti-G12V mutant KRAS therapeutic strategy. Citation Format: Xiyue Hu, Randy Singh, Shana Kelley. Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B014.

Specific regulation of BACH1 by the hotspot mutant p53R175H reveals a distinct gain-of-function mechanism.

Although the gain of function (GOF) of p53 mutants is well recognized, it remains unclear whether different p53 mutants share the same cofactors to induce GOFs. In a proteomic screen, we identified BACH1 as a cellular factor that recognizes the p53 DNA-binding domain depending on its mutation status. BACH1 strongly interacts with p53R175H but fails to effectively bind wild-type p53 or other hotspot mutants in vivo for functional regulation. Notably, p53R175H acts as a repressor for ferroptosis by abrogating BACH1-mediated downregulation of SLC7A11 to enhance tumor growth; conversely, p53R175H promotes BACH1-dependent tumor metastasis by upregulating expression of pro-metastatic targets. Mechanistically, p53R175H-mediated bidirectional regulation of BACH1 function is dependent on its ability to recruit the histone demethylase LSD2 to target promoters and differentially modulate transcription. These data demonstrate that BACH1 acts as a unique partner for p53R175H in executing its specific GOFs and suggest that different p53 mutants induce their GOFs through distinct mechanisms.

Prospective Identification of Prognostic Hot-Spot Mutant Gene Signatures for Leukemia: A Computational Study Based on Integrative Analysis of TCGA and cBioPortal Data.

The advantage of an increasing amount of bioinformatics data on leukemias intrigued us to explore the hot-spot mutation profiles and investigate the implications of those hot-spot mutations in patient survival. We retrieved somatic mutations and their distribution in protein domains through data analysis of The Cancer Genome Atlas and cBioPortal databases. After determining differentially expressed mutant genes related to leukemia, we further conducted principal component analysis and single-factor Cox regression analyses. Moreover, survival analysis was performed for the obtained candidate genes, followed by a multi-factor Cox proportional hazard model method for the impacts of the candidate genes on the survival and prognosis of patients with leukemia. At last, the signaling pathways involved in leukemia were investigated by gene set enrichment analysis. There were 223 somatic missense mutation hot-spots identified with pertinence to leukemia, which were distributed in 41 genes. Differential expression in leukemia was witnessed in 39 genes. We found a close correlation between seven genes and the prognosis of leukemia patients, among which, three genes could significantly influence the survival rate. In addition, among these three genes, CD74 and P2RY8 were highlighted due to close pertinence with survival conditions of leukemia patients. Finally, data suggested that B cell receptor, Hedgehog, and TGF-beta signaling pathways were enriched in low-hazard patients. In conclusion, these data underline the involvement of hot-spot mutations of CD74 and P2RY8 genes in survival status of leukemia patients, highlighting their as novel therapeutic targets or prognostic indicators for leukemia patients. Summary of Graphical Abstract: We identified 223 leukemia-associated somatic missense mutation hotspots concentrated in 41 different genes from 2297 leukemia patients in the TCGA database. Differential analysis of leukemic and normal samples from the TCGA and GTEx databases revealed that 39 of these 41 genes showed significant differential expression in leukemia. These 39 genes were subjected to PCA analysis, univariate Cox analysis, survival analysis, multivariate Cox regression analysis, GSEA pathway enrichment analysis, and then the association with leukemia survival prognosis and related pathways were investigated.

Predicting protein-membrane interfaces using molecular simulations and ensemble machine learning.

Abnormal protein-membrane attachment is involved in deregulated cellular pathways and in disease. The possibility to modulate protein-membrane interactions represents a new promising therapeutic strategy for membrane proteins. In this talk, we explore the free energy landscape of membrane protein dimerization using parallel tempering metadynamics simulations in the well-tempered ensemble and coarse-grained force fields and reproduce the structure and energetics of the dimerization process of membrane proteins and proteins in an aqueous solution in reasonable accuracy and throughput. We propose that the use of enhanced sampling simulations with a refined coarse-grained force field and appropriately defined collective variables is a robust approach for studying the protein dimerization process, although one should be cautious of the energy minima ranking. Moreover, we study oncogenes, including the H1047R and E545K hotspot mutants of PI3Kα, and KRAS-4B to understand the basis of protein overactivation. We calculate their allosteric pathways and show residues important in delivering communication signals between functional domains of each protein. Finally, we describe an ensemble machine learning methodology to predict protein-membrane interfaces of peripheral membrane proteins and present a drug design pipeline for drugging protein-membrane interfaces using the DREAMM web-server https://dreamm.ni4os.eu. Taking into account these results, we investigate opportunities for allosteric drug design.

A multi-population-based genomic analysis uncovers unique haplotype variants and crucial mutant genes in SARS-CoV-2

BackgroundCOVID-19 is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Rigorous detection and treatment strategies against SARS-CoV-2 have become very challenging due to continuous evolutions to the viral genome. Therefore, careful genomic analysis is sorely needed to understand transmission, the cellular mechanism of pathogenicity, and the development of vaccines or drugs. ObjectiveIn this study, we intended to identify SARS-CoV-2 genome variants that may help understand the cellular and molecular foundation of coronavirus infections required to develop effective intervention strategies. MethodsSARS-CoV-2 genome sequences were downloaded from an open-source public database, processed, and analyzed for variants in target detection sites and genes. ResultsWe have identified six unique variants, G---AAC, T---AAC---T, AAC---T, AAC--------T, C----------T, and C--------C, at the nucleocapsid region and eleven major hotspot mutant genes: nsp3, surface glycoprotein, nucleocapsid phosphoprotein, ORF8, nsp6, nsp2, nsp4, helicase, membrane glycoprotein, 3′-5′ exonuclease, and 2′-O-ribose methyltransferases. In addition, we have identified eleven major mutant genes that may have a crucial role in SARS-CoV-2 pathogenesis. ConclusionStudying haplotype variants and 11 major mutant genes to understand the mechanism of action of fatal pathogenicity and inter-individual variations in immune responses is inevitable for managing target patient groups with identified variants and developing effective anti-viral drugs and vaccines.

Reduction of Missed Diagnosis of G6PD Deficiency in Heterozygous Females by G6PD/6PGD Ratio Assay Combined with Amplification Refractory Mutation System PCR

Objective: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder that results in impaired enzyme activity. The G6PD/6PGD ratio assay was routinely used for G6PD deficiency screening in China, but there is an apparent defect of missed diagnosis in heterozygous females. The study aims to explore the means to improve its accuracy. Methods: A total of 4,161 Chinese females of childbearing age were collected in this retrospective study. All samples were first subjected to G6PD/6PGD ratio assay and then screened by amplification refractory mutation system PCR (ARMS-PCR) for six hotspot mutants in Chinese population (c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, c.392G>T, and c.871G>A). For the samples with G6PD/6PGD ratio<1.0 and no mutations were found by ARMS-PCR, next-generation sequencing (NGS) was performed. Sanger sequencing was finally used to verify all the variants. Results: The prevalence of G6PD deficiency in Shenzhen females of childbearing age was 7.31%. The proportion of the six hotspot mutations accounted for 98.03% of all 304 G6PD variants carriers. Taking the ARMS-PCR/NGS results as a reference, the missed diagnosis rate of the G6PD/6PGD ratio assay was 33.88%. Using ARMS-PCR to retest the samples with a G6PD/6PGD ratio between 1.00 and ∼1.10 or 1.00 and ∼1.15 could reduce the missed diagnosis rate from the original 33.88% to 18.09% or 12.05% separately. Conclusion: ARMS-PCR is an appropriate supplementary method for discovering most carriers missed by the G6PD/6PGD ratio assay.

Mutant p53K120R expression enables a partial capacity to modulate metabolism.

The TP53 tumor suppressor gene is one of the most studied gene in virtue of its ability to prevent cancer development by regulating apoptosis, cell cycle arrest, DNA repair, autophagy and senescence. Furthermore, the modulation of metabolism by P53 is fundamental for tumor suppressor activity. Studies in mouse models showed that mice carrying TP53 mutations affecting the acetylation in the DNA binding domain still retain the ability to transactivate genes involved in metabolism. Noteworthy, mice expressing the triple 3KR or the single K117R mutant do not show early on-set tumor development in contrast to TP53 −/− mice. Interestingly, the mouse K117R mutation corresponds to the human tumor-derived K120R modification, which abrogates P53-dependent activation of apoptosis without affecting growth arrest. In this study, we investigated the property of the human P53 K120R mutant in the regulation of metabolism by analyzing the transcriptional specificity in yeast- and mammalian-based reporter assays, the metabolic phenotype associated to its expression in colon cancer HCT116 TP53−/− cells and the induction of P53 targets and proteins involved in the antioxidant response. These properties were analyzed in comparison to wild type P53 protein, the human triple mutant corresponding to mouse 3KR and the cancer hot-spot R273H mutant. We confirm the selective functionality of P53 K120R mutant, which shows a transcriptional activity on cell cycle arrest but not on apoptotic targets. Interestingly, this mutant shows a partial transactivation activity on p53 response element belonging to the metabolic target TIGAR. Moreover, we observe a significant uncoupling between oxygen consumption and ATP production associated with higher lipid peroxidation level in all P53 mutants carrying cells with respect to wild type P53 expressing cells. Noteworthy, in the absence of a pro-oxidative challenge, cells expressing K120R mutant retain a partial capacity to modulate glucose metabolism, limiting lipid peroxidation with respect to the other P53 mutants carrying cells. Lastly, especially in presence of human 3KR mutant, a high expression of proteins involved in the antioxidant response is found. However, this response does not avoid the increased lipid peroxidation, confirming that only wild type P53 is able to completely counteract the oxidative stress and relative damages.

Chemoselective Covalent Modification of K-Ras(G12R) with a Small Molecule Electrophile.

KRAS mutations are one of the most common oncogenic drivers in human cancer. While small molecule inhibitors for the G12C mutant have been successfully developed, allele-specific inhibition for other KRAS hotspot mutants remains challenging. Here we report the discovery of covalent chemical ligands for the common oncogenic mutant K-Ras(G12R). These ligands bind in the Switch II pocket and irreversibly react with the mutant arginine residue. An X-ray crystal structure reveals an imidazolium condensation product formed between the α,β-diketoamide ligand and the ε- and η-nitrogens of arginine 12. Our results show that arginine residues can be selectively targeted with small molecule electrophiles despite their weak nucleophilicity and provide the basis for the development of mutant-specific therapies for K-Ras(G12R)-driven cancer.

Chk1 inhibition-induced BRCAness synergizes with olaparib in p53-deficient cancer cells

ABSTRACT Although targeting DNA-damage repair by inhibition of PARP exhibits weak or modest single-agent activity due to the existence of functional BRCA1/2 alleles, PARP inhibitors have been gradually applicable in BRCA-proficient cancers. Checkpoint kinase 1 (Chk1) inhibition selectively disrupts homologous recombination (HR)-mediated DNA repair and confers synthetic lethality in p53-deficient tumors, we therefore aim at expounding the chemopotentiating effects of Chk1 inhibition on PARPi in BRCA-proficient and p53-deficient cancer cells. Initially, BRCA wild-type, p53-null cells including AsPC-1 and H1299 demonstrated innate resistance to PARP inhibitor olaparib compared to BRCA1-mutant, p53-null MDA-MB-436 cells. We quantified the interaction between olaparib and a selective Chk1 inhibitor MK-8776, which produced synergistic effects under sub-IC50 concentrations in p53-depleted AsPC-1 and H1299 cells. Olaparib in combination with MK-8776 showed enhanced antitumor effects through prohibiting proliferation and secondarily inducing apoptosis in two cell lines. Of note, we observed that MK-8776 significantly sensitized cells to olaparib by broad DNA and chromosomal breaks. Mechanistically, MK-8776 abrogated olaparib-induced BRCA1 intranuclear foci formation, MCM7-mediated replication machineries, and ultimately triggered an accumulation of γH2AX, a well-recognized marker of DNA double-strand breaks. Additionally, we established ectopic expression of hotspot mutant p53 in H1299 cells. Introduction of p53R175 H promoted olaparib resistance as single-agent treatment, but the synergy between olaparib and MK-8776 was still achievable and the region of synergy was produced by lower combination concentrations. These data provide insight into how Chk1 inhibition could be effectively targeted and confer sensitivity to olaparib toward p53-deficient and HR-proficient cancers.

Discovery of compounds that reactivate p53 mutants in vitro and in vivo

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted invitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.

Chemical acylation of an acquired serine suppresses oncogenic signaling of K-Ras(G12S)

Drugs that directly impede the function of driver oncogenes offer exceptional efficacy and a therapeutic window. The recently approved mutant selective small-molecule cysteine-reactive covalent inhibitor of the G12C mutant of K-Ras, sotorasib, provides a case in point. KRAS is the most frequently mutated proto-oncogene in human cancer, yet despite success targeting the G12C allele, targeted therapy for other hotspot mutants of KRAS has not been described. Here we report the discovery of small molecules that covalently target a G12S somatic mutation in K-Ras and suppress its oncogenic signaling. We show that these molecules are active in cells expressing K-Ras(G12S) but spare the wild-type protein. Our results provide a path to targeting a second somatic mutation in the oncogene KRAS by overcoming the weak nucleophilicity of an acquired serine residue. The chemistry we describe may serve as a basis for the selective targeting of other unactivated serines.