Biology subjects

Zafra, M. P.

Publications and source records attributed to Zafra, M. P..

6 recordsLinked to original sources

bioRxiv2025

Molecular dynamics driving phenotypic divergence among KRAS mutants in pancreatic tumorigenesis

Inflammation in the pancreas drives acinar-to-ductal metaplasia (ADM), a progenitor-like state that can be hijacked by mutant Kras in the formation of pancreatic cancer (PDAC). How these cell fate decisions vary according to KRAS mutation remains poorly understood. To define mutation-specific lineage reversion and tumor initiation, we implement novel Ptf1a-TdTomato mice and multiple KRAS mutants across an array of genetic, pharmacologic, and inflammatory perturbations in vivo. Whereas KRASG12D co-opts injury to enable lineage reversion, enhancer reprogramming, and tumor initiation, KRASG12R/V can initiate but not sustain dedifferentiated and neoplastic transcriptional and epigenetic programs. We find the KRASG12R/V defects consist of a failure to invoke robust EGFR signaling and activate Rac1/Vav1, with constitutive Akt activation in vivo sufficient to rescue the tumorigenic potential of KRASG12R. As the marked heterogeneity among KRAS variants begins early in tumorigenesis, these data are crucial to understanding mutation-specific oncogenic trajectories and directing the implementation of KRAS-directed therapeutics. SIGNIFICANCEDefining how KRAS mutants drive distinct outcomes in human pancreatic cancer is critical for developing allele-specific therapeutic approaches. This study unveils a hierarchy among KRASG12D, KRASG12V, and KRASG12R to drive tumor initiation, owing to heterogeneous activation of EGFR, PI3K/AKT, and RAC1 signaling, thus revealing mutation-specific evolutionary paths in pancreatic tumorigenesis.

cancer biology↗

bioRxiv2022

Rapid generation of precision preclinical cancer models using regulatable in vivo base editing

Single nucleotide variants (SNVs) comprise the majority of cancer-associated genetic changes and can have diverse effects on protein function. Despite a comprehensive catalogue of SNVs across human cancers, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a murine system for temporal and regulatable in vivo cytosine base editing (iBE). The iBE mice show robust, doxycycline-dependent expression across a broad range of tissues with no evidence of DNA or RNA off-target effects. Transient iBE induction drives efficient creation of individual or multiple SNVs in intestinal, lung, and pancreatic organoids, while temporal iBE regulation allows controlled sequential genome editing. Moreover, in situ delivery of plasmid-based or synthetic sgRNAs to target tissues facilitates the simple and rapid generation of pre-clinical cancer models. Overall, iBE is a powerful in vivo platform to define and interrogate the genetic drivers of cancer.

cancer biology↗

bioRxiv2020

Lineage reversion drives WNT independence in intestinal cancer

The WNT pathway is a fundamental regulator of intestinal homeostasis and hyperactivation of WNT signaling is the major oncogenic driver in colorectal cancer (CRC). To date, there are no described mechanisms that bypass WNT dependence in intestinal tumors. Here, we show that while WNT suppression blocks tumor growth in most organoid and in vivo CRC models, the accumulation of CRC-associated genetic alterations enables drug resistance and WNT-independent growth. In intestinal epithelial cells harboring mutations in KRAS or BRAF, together with disruption of p53 and SMAD4, transient TGF{beta} exposure drives YAP/ TAZ-dependent transcriptional reprogramming and lineage reversion. Acquisition of embryonic intestinal identity is accompanied by a permanent loss of adult intestinal lineages, and long-term WNT-independent growth. This work delineates genetic and microenvironmental factors that drive WNT inhibitor resistance, identifies a new mechanism for WNT-independent CRC growth and reveals how integration of associated genetic alterations and extracellular signals can overcome lineage-dependent oncogenic programs.

cancer biology↗

bioRxiv2019

GO: A functional reporter system to identify and enrich base editing activity

Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids, and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a "Gene On" (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency, and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes, or enzymes such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.

bioengineering↗

bioRxiv2019

An in vivo KRAS allelic series reveals distinct phenotypes of common oncogenic variants

KRAS is the most frequently mutated oncogene in cancer. Tumor sequencing has revealed a complex spectrum of KRAS mutations across different cancer types, yet there is little understanding how specific KRAS alterations impact tumor in initiation, progression, or therapy response. Using high-fidelity CRISPR-based engineering, we created an allelic series of new LSL-Kras mutant mice, reflecting codon 12 and 13 mutations that are highly prevalent in lung (KRASG12C), pancreas (KRASG12R) and colon (KRASG13D) cancers. Induction of each mutation in the developing mouse pancreas reveal striking quantitative and qualitative differences in the degree of ductal transformation and pre-malignant progression. Further, using organoid models we show that KRASG13D mutants respond to EGFR inhibition, while the anti-proliferative effect of KRASG12C-selective inhibitors can be overcome by upstream EGFR signaling. Together, these new mouse strains provide an ideal for investigating KRAS biology in vivo, and for developing pre-clinical precision oncology models of KRAS-mutant pancreas (G12R), colon (G13D), and lung (G12C) cancers.

cancer biology↗

bioRxiv2018

An optimized toolkit for precision base editing

CRISPR base editing is a potentially powerful technology that enables the creation of genetic mutations with single base pair resolution. By re-engineering both DNA and protein sequences, we developed a collection of constitutive and inducible base editing vector systems that dramatically improve the ease and efficiency by which single nucleotide variants can be created. This new toolkit is effective in a wide range of model systems, and provides a means for efficient in vivo somatic base editing.

bioengineering↗