Mutations in the TP53 tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specific TP53 missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position—R273C vs. R273H—has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors.

Abstract Breast tumors present a high degree of heterogeneity. The major tumor suppressor TP53 is mutated in 30% of all breast tumors and 80% in basal subtype co-existing with numerous other somatic mutations. We hypothesized that combination neo-morphic functions of specific TP53 driver mutations and combination of unique ‘co-drivers' result in inter-tumor heterogeneity. We observed a broad spectrum of phenotypic changes in hallmarks of cancer like cell survival, resistance to apoptosis and anoikis, cell migration and invasion among a panel of 10 mutant p53 expressing mammary epithelial cell lines compared to those with WT p53. Integrated analysis from ChIP-seq and RNA-seq revealed distinct promoter binding profiles of the different mutant p53 proteins, implying non-canonical transcriptional activity contributing to the possible phenotypic heterogeneity in TP53-mutated tumors. The phenotypic heterogeneity of seen in TP53 mutated breast tumors can be attributed to both altered DNA binding properties resulting in expression changes of canonical or mutant-specific target genes and functional crosstalk with distinct co-existing somatic mutations or co-drivers. For the proof-of-concept of co-drivers of mutant p53, when PTEN was deleted using CRISPR in non-invasive p53 mutant Y234C expressing cells a remarkable increase in cell invasion was observed. A genome wide CRISPR based pooled library screen maintained at low MOI on two distinct p53 mutant Y234C and R273C cells followed by bulk sequencing identified completely different candidate co-driver mutations that promoted cell invasion, the initiating step for tumor cell metastasis. The top co-driver candidates included unique genes associated with cytoskeletal and apoptosis resistance pathways and some known mutated genes reported in breast cancer patients harboring TP53 mutations. These invasive pooled library transduced cells when injected in a xenograft mouse model failed to form any mammary tumors. Interestingly, primary tumors appeared in mice injected with pooled library cells transduced at high MOI along with overexpression of MYC. Such results reinstated the importance of functional crosstalk between genes and specific gene combinations driving tumorigenesis. We are currently optimizing a single-cell sequencing platform to capture the combinatorial co-drivers of mutant TP53 promoting the mice primary tumors. This combined approach of deep molecular profiling and functional genomic screen powered the identification of distinct mutant TP53 driver and co-driver gene sets that may contribute to heterogeneous cellular phenotypes and promote aggressive behavior which can guide the development of novel targeted therapies. Citation Format: Anasuya Pal, Chenxi Xu, Jin Park, Joshua LaBaer. A genome wide functional genomics screen for co-driver mutations of mutant TP53 promoting cellular heterogeneity during breast cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-036.

Antimicrobial peptides (AMPs) exhibit cell selectivity and activity against microorganisms and are promising candidates as pharmaceutical agents. In mammalian cell membranes cholesterol plays a regulatory function in antibiotic drug resistance and the immune response. Our hypothesis is that differences in the peptide-membrane interactions versus cholesterol affect the bilayer properties giving a possible framework for selectivity of AMPs for bacterial membranes. Here we employed solid-state 2H NMR to compare the degree of softening or stiffening by AMPs compared to cholesterol in model di-monounsaturated phosphatidylcholine and phosphatidylethanolamine lipids (DOPC vs.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks three major drug-targetable receptors, ER, PR, and HER2. TNBC patients have much worse 5-year survival rates (60%) in contrast to 90% for other breast cancer subtypes and display highly heterogeneous molecular profiles, cellular phenotypes, and drug responses, which poses major challenges in patient treatments. The tumor suppressor gene TP53 is mutated in 30% of breast tumors overall and but highly prevalent (~80%) in TNBC. Unlike mutations in other tumor suppressor genes that are predominantly loss-of-function deletions or truncations, TP53 mutations occur mostly as >100 different missense mutations within the DNA binding domain, implying that the mutant proteins may exert both loss-of-function activities and gain of distinct neomorphic functions, thus contributing to phenotypic heterogeneity of TNBC. When we characterized systematically a panel of MCF10A cell lines expressing 10 most prevalent missense mutant p53 proteins, the cell lines indeed displayed highly diverse neomorphic cellular phenotypes distinct from those of p53-knockdown cells. To investigate molecular mechanisms underlying the heterogeneity, we then performed RNA-Seq and pathway analysis and identified the key pathways, such as the Hippo/YAP pathway, that were dysregulated correlatively with phenotypic aggressiveness of the mutant p53 cell lines. In addition, ChIP-Seq analysis revealed that promoter binding capacity and preference of mutant p53 proteins associated with more aggressive phenotypes were more severely affected, especially for the genes in the dysregulated pathways identified from RNA-Seq analysis. These demonstrated collectively that different missense p53 mutations lead to heterogeneous phenotypes by exerting distinct neomorphic molecular functions. Further, given that TP53 mutations by themselves cannot drive full cancer progression, these imply that cells with different p53 missense mutations need distinct sets of additional “co-driver” mutations and alteration of cellular programs specific to each mutation for full cancer progression, representing potential molecular targets for personalized therapies. Supporting this hypothesis, when we performed genome-wide in vitro CRISPR screens in search of co-driver mutations specific to different p53 mutations, a unique set of hits was identified for each mutant p53-expressing cell lines. However, in in vivo mouse xenograft models, even the cells expressing aggressive p53 mutants such R273C failed to develop tumors upon transducing gene-deleting CRISPR gRNA libraries at high MOI. Based on reasoning that development of tumor requires mutations in both tumor suppressors and oncogenes, we then performed CRISPR screens on the p53-R273C cells overexpressing MYC, a known oncogene for TNBC, and observed tumor formation within 9 weeks, only after the CRISPR library transduction. By next-generation sequencing of the gRNA cassettes amplified from the tumors, >20 novel co-driver candidates in addition to known tumor suppressors such as NF2 and PTEN were identified. Interestingly, ARAF, a proto-oncogene, was one of the top candidates found in multiple tumors, and the targeted sequencing confirmed out-of-frame deletions resulting in truncated proteins with only the N-term Ras-binding domain. We are currently validating the functional relevance of these findings in conjunction with the dysregulated pathways identified from RNA-Seq and ChIP-Seq analysis. Taken together, our integrated approach of utilizing phenotyping, multi-omics bioinformatics analysis, and screening has revealed the molecular mechanisms underlying phenotypic and molecular heterogeneity and potential molecular targets of TNBC. Citation Format: Dustin Grief, Anasuya Pal, Laura Gonzalez-Malerva, Seron Eaton, Chenxi Xu, Grant Christensen, Joy Blain, Nicholas Mellor, Jason Steel, Chitrak Gupta, Ellen Streitwieser, Abhishek Singharoy, Jin Park, Joshua LaBaer. A genome-wide functional genomics screen reveals unique co-driver mutations of mutant TP53 promoting cellular heterogeneity during breast cancer progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-05.

Antimicrobial peptides (AMPs) are found in the innate immune systems of most living organisms. These peptides exhibit cell selectivity, and activity against a broad spectrum of microorganisms, making them promising candidates as antimicrobial biomaterials. The AMPs are anchored with polymer tethers and biologically synthesized as functionalized biomaterials. We successfully prepared LL-37 conjugated biopolymer materials with antimicrobial activity, which switch to micelles at temperatures between 27-30 oC. Understanding the peptide-membrane interactions represents the basis for AMP's selectivity for bacterial cell membranes. We hypothesized that peptide insertion is assisted by membrane curvature. Models of a gram-negative bacterial outer membrane comprising POPE/DMPG/CL (90:5:5) and a membrane with the composition DMPC/DMPG/CL (90:5:5) were investigated using molecular dynamics simulations. The antimicrobial peptide LL-37 was arranged on the surface of the membranes as "carpets" of ordered peptides. We also performed all-atom molecular dynamics simulations using NAMD 2.13 to visualize the carpet-to-barrel or toroidal-pore transition. To determine the energetically favorable model, the non-bonded interactions between the carpet and pore models were compared using the NAMD energy. From our in-silico observations, the pore model is more favorable than the carpet model for the same peptide/lipid ratio. Critical values of the peptide/lipid ratio required for cell penetration were investigated to determine the rate of peptide insertion into the membrane with different LL-37 concentrations. From the simulation timescale (in microseconds), as the peptide/lipid ratio increased, partial insertion and membrane curvature were observed in the bacterial mimic model. Further, the C-terminal helix of LL-37 was observed to unfold when interacting with phosphate head groups of the lipids. The propensity for AMPs to insert into the lipid bilayer via the N-terminus or C-terminus was similar. These observations from molecular dynamics simulations provide a basis for designing more advanced functionalized antimicrobial-biomaterials.

Driving molecular dynamics simulations with data-guided collective variables offer a promising strategy to recover thermodynamic information from structure-centric experiments. Here, the three-dimensional electron density of a protein, as it would be determined by cryo-EM or x-ray crystallography, is used to achieve simultaneously free-energy costs of conformational transitions and refined atomic structures. Unlike previous density-driven molecular dynamics methodologies that determine only the best map-model fits, our work employs the recently developed Multi-Map methodology to monitor concerted movements within equilibrium, non-equilibrium, and enhanced sampling simulations. Construction of all-atom ensembles along the chosen values of the Multi-Map variable enables simultaneous estimation of average properties, as well as real-space refinement of the structures contributing to such averages. Using three proteins of increasing size, we demonstrate that biased simulation along the reaction coordinates derived from electron densities can capture conformational transitions between known intermediates. The simulated pathways appear reversible with minimal hysteresis and require only low-resolution density information to guide the transition. The induced transitions also produce estimates for free energy differences that can be directly compared to experimental observables and population distributions. The refined model quality is superior compared to those found in the Protein Data Bank. We find that the best quantitative agreement with experimental free-energy differences is obtained using medium resolution density information coupled to comparatively large structural transitions. Practical considerations for probing the transitions between multiple intermediate density states are also discussed.