The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

Validation of the Alternative International Prognostic Score-E (AIPS-E): Analysis of Binet stage A chronic lymphocytic leukemia patients enrolled into the O-CLL1-GISL protocol.

OBJECTIVES: To validate the predictive value on time to first treatment (TTFT) of AIPS-E and IPS-E evaluated in an independent cohort of newly diagnosed and non-referred Binet stage A CLL patients enrolled in the O-CLL1-GISL protocol (clinicaltrial.gov identifier: NCT00917540). METHODS: A cohort of 292 newly diagnosed Binet A CLL cases has been enrolled in the study. Patients from several Italian Institutions were prospectively enrolled within 12 months of diagnosis into the O-CLL1-GISL protocol. RESULTS: The majority of patients were male (62%); median age was 60.4 years, 102 cases (34.9%) showed unmutated IGHV genes, 8 cases (2.8) the presence of del(11q)/del(17p), 142 cases (48.6%) the presence of palpable lymph nodes and 146 cases (50%) and ALC > 15 × 109 /l. After a median follow-up of 7.2 years, 130 patients underwent treatment. According to the AIPS-E, 96 patients were classified as low-risk, 128 as intermediate-risk, and 68 as high-risk. These groups showed significant differences in terms of TTFT. The C-statistic was 0.71 (P < .0001) for predicting TTFT. According to IPS-E, 77 patients were classified as low-risk, 135 as intermediate-risk, and 80 as high-risk. These groups showed significant differences in terms of TTFT. The C-statistic was 0.705 (P < .0001) for predicting TTFT. CONCLUSIONS: Our data confirm an accurate prognostic utility of both AIPS-E and IPS-E at the individual patient level. These data may be useful for a precise stratification of early-stage patients.

Comparison of ibrutinib and idelalisib plus rituximab in real-life relapsed/resistant chronic lymphocytic leukemia cases.

OBJECTIVES: To compare the capacity of ibrutinib (IB) and idelalisib-rituximab (IDELA-R) of prolonging overall survival (OS) as in CLL patients, previously treated with chemotherapy only. METHODS: A real-life cohort of 675 cases has been identified and investigated in the database of the groups participating in the study. RESULTS: At an unadjusted univariate analysis, a significant death risk reduction was observed favoring IB (IDELA-R vs IB HR = 0.5, 95% CI = 0.36-0.71) although with some limitations due to the non-randomized and retrospective nature of the study and to the lower number of patients in the IDELA-R group (112 cases) related to the current prescribing practice. To overcome the potential problem of confounding by indication, we adjusted the association between the type of therapy and mortality for all variables significantly associated with OS at Cox univariate analysis. Furthermore, those variables, differently distributed between the two study groups, were introduced into the multivariate Cox model to improve the effectiveness of the analysis. By introducing all these variables into the multiple Cox regression model, we confirmed the protective effect of IB vs IDELA-R (HR = 0.67, 95% CI = 0.45-0.98, P = .04) independent of potential confounders. CONCLUSIONS: Although our analysis presents some constraints, that is, the unavailability of additional potential confounders, and the retrospective nature of the study, this observation may be of help for the daily clinical practice, particularly in the absence of randomized trials comparing the two schedules.

Gambogic acid counteracts mutant p53 stability by inducing autophagy.

Mutant p53 (mutp53) proteins are frequently present at higher levels than the wild-type (wt) protein in tumors, and some of them can acquire oncogenic properties. Consistently, knockdown of mutp53 protein in human cancer cell lines leads to reduced cell proliferation and invasion as well as to an increased sensitivity to some anticancer drugs. Therefore, the exploitation of cellular pathways and/or molecules that promote mutp53 degradation may have a therapeutic interest. Recently, autophagy is emerging as an important pathway involved in the stability of mutp53. In this paper, we explored the autophagic potential of gambogic acid (GA), a molecule that stimulates the degradation of mutp53 and increases the sensitivity of cancer cells to chemotherapeutic agents. We demonstrated that GA may induce mutp53 degradation through autophagy in cancer cells expressing the p53-R280K (MDA-MB-231) and the p53-S241F (DLD1) proteins. The inhibition of autophagy with bafilomycin A1 or chloroquine counteracted mutp53 degradation by GA. However, the autophagy induction and mutp53 degradation affected cell survival and proliferation only at low GA concentrations. At higher GA concentrations, when cells undergo massive apoptosis, autophagy is no longer detectable by immuno-fluorescence analysis. We concluded that autophagy is a relevant pathway for mutp53 degradation in cancer cells but it contributes only partially to GA-induced cell death, in a time and dose-dependent manner.

Human transcription factors in yeast: the fruitful examples of P53 and NF-кB.

The observation that human transcription factors (TFs) can function when expressed in yeast cells has stimulated the development of various functional assays to investigate (i) the role of binding site sequences (herein referred to as response elements, REs) in transactivation specificity, (ii) the impact of polymorphic nucleotide variants on transactivation potential, (iii) the functional consequences of mutations in TFs and (iv) the impact of cofactors or small molecules. These approaches have found applications in basic as well as applied research, including the identification and the characterisation of mutant TF alleles from clinical samples. The ease of genome editing of yeast cells and the availability of regulated systems for ectopic protein expression enabled the development of quantitative reporter systems, integrated at a chosen chromosomal locus in isogenic yeast strains that differ only at the level of a specific RE targeted by a TF or for the expression of distinct TF alleles. In many cases, these assays were proven predictive of results in higher eukaryotes. The potential to work in small volume formats and the availability of yeast strains with modified chemical uptake have enhanced the scalability of these approaches. Next to well-established one-, two-, three-hybrid assays, the functional assays with non-chimeric human TFs enrich the palette of opportunities for functional characterisation. We review ∼25 years of research on human sequence-specific TFs expressed in yeast, with an emphasis on the P53 and NF-кB family of proteins, highlighting outcomes, advantages, challenges and limitations of these heterologous assays.

Studying p53 family proteins in yeast: induction of autophagic cell death and modulation by interactors and small molecules.

In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions.

The TP53 website: an integrative resource centre for the TP53 mutation database and TP53 mutant analysis.

A novel resource centre for TP53 mutations and mutants has been developed (http://p53.fr). TP53 gene dysfunction can be found in the majority of human cancer types. The potential use of TP53 mutation as a biomarker for clinical studies or exposome analysis has led to the publication of thousands of reports describing the TP53 gene status in >10,000 tumours. The UMD TP53 mutation database was created in 1990 and has been regularly updated. The 2012 release of the database has been carefully curated, and all suspicious reports have been eliminated. It is available either as a flat file that can be easily manipulated or as novel multi-platform analytical software that has been designed to analyse various aspects of TP53 mutations. Several tools to ascertain TP53 mutations are also available for download. We have developed TP53MULTLoad, a manually curated database providing comprehensive details on the properties of 2549 missense TP53 mutants. More than 100,000 entries have been arranged in 39 different activity fields, such as change of transactivation on various promoters, apoptosis or growth arrest. For several hot spot mutants, multiple gain of function activities are also included. The database can be easily browsed via a graphical user interface.

Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code.

Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein-DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.

Structural studies on mechanisms to activate mutant p53.

The design of a broad-spectrum cancer drug would provide enormous clinical benefits to treat cancer patients. Most of cancerous cells have a mutation in the p53 gene that results in an inactive mutant p53 protein. For this reason, p53 is a prime target for the development of a broad-spectrum cancer drug. To provide the atomic information to rationally design a drug to recover p53 activity is the main goal of the structural studies on mutant p53. We review three mechanisms that influence p53 activity and provide information about how reactivation of mutant p53 can be achieved: stabilization of the active conformation of the DNA-binding domain of the protein, suppression of missense mutations in the DNA-binding domain by a second-site mutation, and increased transactivation.

Structure of p73 DNA-binding domain tetramer modulates p73 transactivation.

The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation.

TP53 mutants in the tower of babel of cancer progression.

Loss-of-function, partial-function, altered-function, dominant-negative, temperature sensitive, interfering, contact, structural, unfolded, misfolded, dimeric, monomeric, non-cooperative, unstable, supertrans, superstable, intragenic suppressor. TP53 mutants are many, more than 2,000 in fact, and they can be very diverse. Sporadic; germline; gain-of-function (GoF); oncogenic; rebel-angel; yin and yang; prion-like; metastasis-inducer; mediator of chemo-resistance; modifier of stemness. TP53 mutants can impact important cancer clinical variables, in multiple, often subtle ways, as revealed by cell-based assays as well as animal models. Here, we review studies investigating TP53 mutants for their effect on sequence-specific transactivation function, and especially recent findings on how TP53 mutants can exhibit GoF properties. We also review reports on TP53 mutants' impact on cancer cell transcriptomes and studies with Li-Fraumeni patients trying to classify and predict phenotypes in relation to experimentally determined transcription fingerprints. Finally, we provide an example of the complexity of correlating TP53 mutant functionality to clinical variables in sporadic cancer patients. Conflicting results and limitations of experimental approaches notwithstanding, the study of TP53 mutants has provided a rich body of knowledge, mostly available in the public domain and accessible through databases, which is beginning to impact cancer intervention strategies.

PRIMA-1 induces autophagy in cancer cells carrying mutant or wild type p53.

PRIMA-1 is a chemical compound identified as a growth suppressor of tumor cells expressing mutant p53. We previously found that in the MDA-MB-231 cell line expressing high level of the mutant p53-R280K protein, PRIMA-1 induced p53 ubiquitination and degradation associated to cell death. In this study, we investigated the ability of PRIMA-1 to induce autophagy in cancer cells. In MDA-MB-231 and HCT116 cells, expressing mutant or wild type p53, respectively, autophagy occurred following exposure to PRIMA-1, as shown by acridine orange staining, anti-LC3 immunofluorescence and immunoblots, as well as by electron microscopy. Autophagy was triggered also in the derivative cell lines knocked-down for p53, although to a different extent than in the parental cells expressing mutant or wild type p53. In particular, while wild type p53 limited PRIMA-1 induced autophagy, mutant p53 conversely promoted autophagy, thus sustaining cell viability following PRIMA-1 treatment. Therefore, the autophagic potential of PRIMA-1, besides being cell context dependent, could be modulated in a different way by the presence of wild type or mutant p53. Furthermore, since both cell lines lacking p53 were more sensitive to the cytotoxic effect of PRIMA-1 than the parental ones, our findings suggest that a deregulated autophagy may favor cell death induced by this drug.

Comparison of the biological effects of MMS and Me-lex, a minor groove methylating agent: clarifying the role of N3-methyladenine.

N3-methyladenine (3-mA), generated by the reaction of methylating agents with DNA, is considered a highly toxic but weakly mutagenic lesion. However, due to its intrinsic instability, some of the biological effects of the adduct can result from the formation of the corresponding depurination product [an apurinic (AP)-site]. Previously, we exploited Me-lex, i.e. {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2 carboxamido}propane, a minor groove equilibrium binder with selectivity for A/T rich sequences that efficiently reacts with DNA to afford 3-mA as the dominant product, to probe the biology of this lesion. Using human p53 cDNA as a target in a yeast system, a weak increase in mutagenicity was observed in the absence of Mag1 (3-methyladenine-DNA glycosylase 1, mag1), the enzyme devoted to remove 3-mA from DNA. Moreover, a significant increase in mutagenicity occurred in the absence of the enzymes involved in the repair of AP-sites (AP endonucleases 1 and 2, apn1apn2). Since methyl methanesulfonate (MMS) has been extensively used to explore the biological effects of 3-mA, even though it produces 3-mA in low relative yield, we compared the toxicity and mutagenicity induced by MMS and Me-lex in yeast. A mutagenesis reporter plasmid was damaged in vitro by MMS and then transformed into wild-type and Translesion Synthesis (TLS) Polζ (REV3) and Polη (RAD30) deficient strains. Furthermore, a mag1rad30 double mutant strain was constructed and transformed with the DNA plasmid damaged in vitro by Me-lex. The results confirm the important role of Polζ in the mutagenic bypass of MMS and Me-lex induced lesions, with Polη contributing only towards the bypass of Me-lex induced lesions, mainly in an error-free way. Previous and present results point towards the involvement of AP-sites, derived from the depurination of 3-mA, in the observed toxicity and mutagenicity.

EEC- and ADULT-associated TP63 mutations exhibit functional heterogeneity toward P63 responsive sequences.

TP63 germ-line mutations are responsible for a group of human ectodermal dysplasia syndromes, underlining the key role of P63 in the development of ectoderm-derived tissues. Here, we report the identification of two TP63 alleles, G134V (p.Gly173Val) and insR155 (p.Thr193_Tyr194insArg), associated to ADULT and EEC syndromes, respectively. These alleles, along with previously identified G134D (p.Gly173Asp) and R204W (p.Arg243Trp), were functionally characterized in yeast, studied in a mammalian cell line and modeled based on the crystal structure of the P63 DNA-binding domain. Although the p.Arg243Trp mutant showed both complete loss of transactivation function and ability to interfere over wild-type P63, the impact of p.Gly173Asp, p.Gly173Val, and p.Thr193_Tyr194insArg varied depending on the response element (RE) tested. Interestingly, p.Gly173Asp and p.Gly173Val mutants were characterized by a severe defect in transactivation along with interfering ability on two DN-P63α-specific REs derived from genes closely related to the clinical manifestations of the TP63-associated syndromes, namely PERP and COL18A1. The modeling of the mutations supported the distinct functional effect of each mutant. The present results highlight the importance of integrating different functional endpoints that take in account the features of P63 proteins' target sequences to examine the impact of TP63 mutations and the associated clinical variability.

The fading guardian: clinical relevance of TP53 null mutation in high-grade serous ovarian cancers.

Background: we evaluated the concordance between immunohistochemical p53 staining and TP53 mutations in a series of HGSOC. Moreover, we searched for prognostic differences between p53 overexpression and null expression groups. Methods: patients affected by HGSOC were included. For each case p53 immunohistochemical staining and molecular assay (Sanger sequencing) were performed. Kaplan-Meier survival analyses were undertaken to determine whether the type of TP53 mutation, or p53 staining pattern influenced overall survival (OS) and progression free survival (PFS). Results: 34 HGSOC were considered. All cases with a null immunohistochemical p53 expression (n=16) showed TP53 mutations (n=9 nonsense, n=4 in-frame deletion, n=2 splice, n=1 in-frame insertion). 16 out of 18 cases with p53 overexpression showed TP53 missense mutation. Follow up data were available for 33 out of 34 cases (median follow up time 15 month). We observed a significant reduction of OS in p53 null group [HR = 3.64, 95% CI 1.01-13.16]. Conclusion: immunohistochemical assay is a reliable surrogate for TP53 mutations in most cases. Despite the small cohort and the limited median follow up, we can infer that HGSOC harboring p53 null mutations are a more aggressive subgroup.

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.