Acquisition of aneuploidy drives mutant p53-associated gain-of-function phenotypes

Abstractp53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.

Allele-specific suppression in C. elegans reveals details of EMS mutagenesis and a possible moonlighting interaction between the vesicular acetylcholine transporter and ERD2 receptors

A missense mutant, unc-17(e245), which affects the Caenorhabditis elegans vesicular acetylcholine transporter UNC-17, has a severe uncoordinated phenotype, allowing efficient selection of dominant suppressors that revert this phenotype to wild-type. Such selections permitted isolation of numerous suppressors after EMS (ethyl methanesulfonate) mutagenesis, leading to demonstration of delays in mutation fixation after initial EMS treatment, as has been shown in T4 bacteriophage but not previously in eukaryotes. Three strong dominant extragenic suppressor loci have been defined, all of which act specifically on allele e245, which causes a G347R mutation in UNC-17. Two of the suppressors (sup-1 and sup-8/snb-1) have previously been shown to encode synaptic proteins able to interact directly with UNC-17. We found that the remaining suppressor, sup-2, corresponds to a mutation in erd-2.1, which encodes an endoplasmic reticulum retention protein; sup-2 causes a V186E missense mutation in transmembrane helix 7 of ERD-2.1. The same missense change introduced into the redundant paralogous gene erd-2.2 also suppressed unc-17(e245). Suppression presumably occurred by compensatory charge interactions between transmembrane helices of UNC-17 and ERD-2.1 or ERD-2.2, as previously proposed in work on suppression by SUP-1(G84E) or SUP-8(I97D)/synaptobrevin. erd-2.1(V186E) homozygotes were fully viable, but erd-2.1(V186E); erd-2.2(RNAi) exhibited synthetic lethality (like erd-2.1(RNAi); erd-2.2(RNAi)), indicating that the missense change in ERD-2.1 impairs its normal function in the secretory pathway but may allow it to adopt a novel moonlighting function as an unc-17 suppressor.

Conditional deletion of SMN in cell culture identifies functional SMN alleles

Spinal muscular atrophy (SMA) is caused by mutation or deletion of survival motor neuron 1 (SMN1) and retention of SMN2 leading to SMN protein deficiency. We developed an immortalized mouse embryonic fibroblast (iMEF) line in which full-length wild-type Smn (flwt-Smn) can be conditionally deleted using Cre recombinase. iMEFs lacking flwt-Smn are not viable. We tested the SMA patient SMN1 missense mutation alleles A2G, D44V, A111G, E134K and T274I in these cells to determine which human SMN (huSMN) mutant alleles can function in the absence of flwt-Smn. All missense mutant alleles failed to rescue survival in the conditionally deleted iMEFs. Thus, the function lost by these mutations is essential to cell survival. However, co-expression of two different huSMN missense mutants can rescue iMEF survival and small nuclear ribonucleoprotein (snRNP) assembly, demonstrating intragenic complementation of SMN alleles. In addition, we show that a Smn protein lacking exon 2B can rescue iMEF survival and snRNP assembly in the absence of flwt-Smn, indicating exon 2B is not required for the essential function of Smn. For the first time, using this novel cell line, we can assay the function of SMN alleles in the complete absence of flwt-Smn.

Temperature sensitive Mutant Proteome Profiling: a novel tool for the characterization of the global impact of missense mutations on the proteome

ABSTRACTTemperature sensitive (TS) mutants have been foundational in the characterization of essential genes. However, a high-throughput workflow for characterization of biophysical changes in TS mutants is lacking. Temperature sensitive Mutant Proteome Profiling (TeMPP) is a novel application of mass spectrometry (MS) based thermal proteome profiling (TPP) to characterize effects of missense mutations on protein stability and PPIs. This study characterizes missense mutations in two different subunits of the 26S proteasome on the thermal stability of the proteome at large, revealing distinct mechanistic details that were not obtained using only steady-state transcriptome and proteome analyses. TeMPP is a precise approach to measure changes in missense mutant containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Overall, TeMPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, non-biased fashion.

Compound heterozygous mutations in the luteinizing hormone receptor signal peptide causing 46,XY disorder of sex development

Testosterone production by the fetal testis depends on a functional relationship between hCG and the LH/chorionic gonadotropin receptor (LHCGR). Failure of the receptor to correctly respond to its ligand leads to impaired sexual differentiation in males. A phenotypically female patient with pubertal delay had a 46,XY karyotype and was diagnosed with 46,XY disorder of sex development (DSD). Novel compound heterozygous LHCGR mutations were found in the signal peptide: a duplication p.L10_Q17dup of maternal origin, and a deletion (p.K12_L15del) and a p.L16Q missense mutation of paternal origin. cAMP production was very low for both the deletion and duplication mutations and was halved for the missense mutant. The duplication and missense mutations were both expressed intracellularly, but at very low levels at the cell membrane; they were most likely retained in the endoplasmic reticulum. The deletion mutant had a very limited intracellular expression, indicating impaired biosynthesis. There was reduced expression of all three mutants, which was most marked for the deletion mutation. There was also decreased protein expression of all three mutant receptors. In the deletion mutation, the presence of a lower-molecular-weight band corresponding to LHCGR monomer, probably due to lack of glycosylation, and a lack of bands corresponding to dimers/oligomers suggests absent ER entry. This novel case of 46,XY DSD illustrates how different LHCGR signal peptide mutations led to complete receptor inactivation by separate mechanisms. The study underlines the importance of specific regions of signal peptides and expands the spectrum of LHCGR mutations.

Gain-of-Function (GOF) Mutant p53 as Actionable Therapeutic Target

p53 missense mutant alleles are present in nearly 40% of all human tumors. Such mutated alleles generate aberrant proteins that not only lose their tumor-suppressive functions but also frequently act as driver oncogenes, which promote malignant progression, invasion, metastasis, and chemoresistance, leading to reduced survival in patients and mice. Notably, these oncogenic gain-of-function (GOF) missense mutant p53 proteins (mutp53) are constitutively and tumor-specific stabilised. This stabilisation is one key pre-requisite for their GOF and is largely due to mutp53 protection from the E3 ubiquitin ligases Mdm2 and CHIP by the HSP90/HDAC6 chaperone machinery. Recent mouse models provide convincing evidence that tumors with highly stabilized GOF mutp53 proteins depend on them for growth, maintenance, and metastasis, thus creating exploitable tumor-specific vulnerabilities that markedly increase lifespan if intercepted. This identifies mutp53 as a promising cancer-specific drug target. This review discusses direct mutp53 protein-targeting drug strategies that are currently being developed at various preclinical levels.