The post-doctoral researchers from Memorial Sloan Kettering Cancer Centre (MSK) developed an upfront and coherent way to study copy number alterations (CNA) deletions in the laboratory using the pancreatic and melanoma cancer mouse models. The CRISPR-based method revealed clinically relevant observations into the multifaceted nature of 9p21.3 ommissions that contribute to up to 15% of human tumors.

Introduction

Deciphering the genetic underlining of cancer and associated aberrations is an essential goal for cancer research. Several research studies have been directed to characterize the genetic delineation of single-nucleotide variants (SNVs), which typically act on an ON/OFF mechanism that affects the outcome of a single gene in terms of phenotype and severity effects. Several copy number alterations (CNA) often have been linked with affecting multiple genes at a time. Research in CNA has locally focussed on the known drivers affecting different regions, such as co-gained or co-deleted.

MACHETE
Image Description: MACHETE enables efficient engineering of genomic deletions.
 Image Source: https://doi.org/10.1038/s43018-022-00443-5

The repetitive state of CNAs results in the complex phenotype of tumors and unexplainable severity. The loss of chromosome 9p21.3 is one of the loci deemed to be associated with poor prognosis and is the most common homozygous deletion. The 9p21.3 locus encloses multiple fundamental tumor-suppressor genes (TSG), such as the cell cycle inhibitors CDKN2A (encoding p16INK4a and p14ARF) and CDKN2B (encoding p15INK4b), which together activate the major tumor-suppressive pathways p53 and RB. Hence, considering the implications and direction mediated by the 9p21.3 deletions, the overall understanding needs to be addressed.

The post-doctoral researchers from Memorial Sloan Kettering Cancer Centre (MSK), New York, USA, developed an upfront and coherent method using CRISPR-CAS9 to study copy number alterations (CNA) deletions in laboratory pancreatic and melanoma cancer mouse models for functional study. Implies tumors with 9p21.3 deletions can exhibit altered immune infiltrate and an increase in resistance to immune-checkpoint blockade therapies, suggesting that the locus may also influence immune responses. The team demonstrated a novel tool, i.e., molecular alteration of chromosomes with engineered tandem elements (MACHETE), to assist the overall experimental study in facilitating the understanding of genomic deletions.

The MACHETE methodology was constructed to eliminate the need for cloning components such as donor DNA and PCR amplification methodology. The team demonstrated the MACHETE’s ability to generate efficient results in determining the megabase deletions. Firstly, a cassette encoding negative and positive selection markers collectively are amplified and inserted into the region of interest facilitated by CRISPR homology-directed repair. Subsequently, cells with integrations were complemented by positive selection. Secondly, a pair of single guide RNAs (sgRNAs) targeting the breakpoints of the intended deletion were instigated, which is followed by negative selection. The specificity of the flanking guide sequences allows homozygous deletions across the pan-cancer dataset hence eliminating the integrations that are mishit. 

Researchers demonstrated the utility of MACHETE on both cancer cells. They expanded the applicability by engineering 0.4 and 1.3-Mb deletions of chromosome 4C4 in embryonic stem cells of the murine model, enabling the circumstances of the creation of germline deletion. Hence, MACHETE is a tailor-made approach to efficiently engineer and decipher large chromosomal deletions across various cellular organizations.

Francisco M. Barriga and the research team relatively identified the loss of type I, IFN genes in the 9p21.3 deleted tumors. Earlier research analysis targeting the TCGA dataset demonstrated that 14 different tumor types foster homozygous 9p21.3 deletions in over 10% of tumor cases. The team also classified 9p21.3 deletions into those targeting the CDKN2A/B alone or more significant events that typically enclosed the entire cluster of type I IFN. The analysis concluded the human PDAC (Pancreatic ductal adenocarcinoma) indicates that CDKN2A deletions are often a preliminary event occurring in tumor evolution.

Provided the role of type I IFNs in modulating immunity, researchers established a study to construct 9p deletions in an isogenic mouse model using PDAC derived from the established pancreatic ductal epithelial cells (PDEC). Since PDECs provide a suitable platform for MACHETE-based engineering and genetic analysis of 9p21.3-equivalent deletions. The study of the tumor phenotypes in an immune-competent context is based on an in vitro model that led the team to generate Trp53 knockout PDEC cells using transient CRISPR-Cas9 and instigated an EGFP-luciferase cassette to enable and visualize engrafted cells, i.e., PDEC sgP53-EL cells.

The findings demonstrated that despite the general nature of CNAs across cancers, their functional characterization had been restricted by the difficulty in manipulating large genomic regions. The developed approach addressed these shortcomings, as it is a customizable and efficient method that enables engineering the deletions of at least 45 Mb, and does not require any cloning of the targeting vectors; it eliminates cells with off-target integrations and also allows engineering the allelic series of deletions. The genetic understanding revealed how 9p21.3 specifies the interferon signaling and immune states in human PDAC. Simultaneously, in the context of functional studies, the results demonstrated the downregulation of type I interferon signaling by genetic or different means and also promoting PDAC metastasis.

Final Thoughts

Since mutations in cancer are often referred to as errors and have been associated with the aberrant functioning of genes leading to various human genetic disorders. The current study by the researchers has broken new facets regarding one of the most frequent copy number alterations in human cancers. Since these CNAs cause a tumor burden, adding to the heterogeneity of cancer, the team deciphers the underlined biology using genetic analysis. MACHETE provides a new framework for investigating significant deletion events beyond just cancer. Using

MACHETE researchers have revealed clinically relevant insights into the multifactorial nature of 9p21.3 deletions that affect immune evasion in tumors. The findings have suggested the co-deletion of type I IFN cluster alongside CDKN2A/B and in nearly half of all tumors harboring 9p21.3 deletions. While other than 9p21.3, genes such as Mtap46 also influence tumor behavior. Type I IFNs are critical tumor suppressors validating the results for evasion evident by changes on the genetic level observed in the tumor model. Through the fundamental insights, the MACHETE has demonstrated the clinical implications for the classification of patients receiving ICB (immune checkpoint blockade) therapy, which has well-known heterogeneous patterns of responses.

Article Sources: Reference Paper

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Mahi Sharma is a consulting Content Writing Intern at the Centre of Bioinformatics Research and Technology (CBIRT). She is a postgraduate with a Master's in Immunology degree from Amity University, Noida. She has interned at CSIR-Institute of Microbial Technology, working on human and environmental microbiomes. She actively promotes research on microcosmos and science communication through her personal blog.

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