Biology subjects

Teh, S. S. K.

Publications and source records attributed to Teh, S. S. K..

3 recordsLinked to original sources

bioRxiv2024

Islands of genomic stability in the face of genetically unstable metastatic cancer

AbstractO_ST_ABSIntroductionC_ST_ABSMetastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here we test the hypothesis that metastatic cancer can be genetically targeted by exploiting single base substitution mutations unique to individual cells that occur as part of normal aging prior to transformation. These mutations are targetable because [~]10% of them form novel tumor-specific "NGG" protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9. MethodsWhole genome sequencing was performed on five rapid autopsy cases of patient-matched primary tumor, normal and metastatic tissue from pancreatic ductal adenocarcinoma decedents. CRISPR-Cas9 PAM targets were determined by bioinformatic tumor-normal subtraction for each patient and verified in metastatic samples by high-depth capture-based sequencing. ResultsWe found that 90% of PAM targets were maintained between primary carcinomas and metastases overall. We identified rules that predict PAM loss or retention, where PAMs located in heterozygous regions in the primary tumor can be lost in metastases (private LOH), but PAMs occurring in regions of loss of heterozygosity (LOH) in the primary tumor were universally conserved in metastases. ConclusionsRegions of truncal LOH are strongly retained in the presence of genetic instability, and therefore represent genetic vulnerabilities in pancreatic adenocarcinomas. A CRISPR-based gene therapy approach targeting these regions may be a novel way to genetically target metastatic cancer.

cancer biology↗

bioRxiv2023

Somatic protospacer adjacent motifs are numerous and selectively targetable in cancers

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within the noncoding regions. We propose a novel, cancer-specific killing approach using CRISPR-Cas9 which exploits the requirement of a protospacer adjacent motif (PAM) for Cas9 activity. Through whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002), we identified an average of 417 somatic PAMs per tumor produced from single base substitutions. We analyzed 591 paired T-N samples from The International Cancer Genome Consortium and discovered medians of [~]455 somatic PAMs per tumor in pancreatic, [~]2800 in lung, and [~]3200 in esophageal cancer cohorts. Finally, we demonstrated >80% selective cell death of two targeted pancreatic cancer cell lines in co-cultures using 4-9 sgRNAs, targeting noncoding regions, designed from the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs through WGS. Statement of significanceThis study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy. It requires just a few targets to induce double strand breaks for significant cytotoxicity. Our findings markedly expand the repertoire of targetable mutations in cancers and support genetically targeting other adult solid tumor types.

cancer biology↗

bioRxiv2023

Mechanism of delayed cell death following simultaneous CRISPR-Cas9 targeting in pancreatic cancers

While radiation is an effective oncologic therapy, killing cancer by inducing DNA double-strand breaks (DSBs), it lacks specificity for neoplastic cells. We have previously adapted the CRISPR-Cas9 gene-editing technology as a cancer-specific treatment modality targeting somatic mutations in pancreatic cancer (PC). However, its tumoricidal potential remains unclear, especially in comparison to therapeutic doses of radiation. Here, we demonstrate that CRISPR-Cas9-induced DSBs are more cytotoxic in PCs than a comparable number of radiation-induced DSBs. We observed >90% tumor growth inhibition by targeting 9 sites with cancer-specific single-guide RNAs (sgRNAs). Through both bioinformatics and cytogenetics analyses, we found that CRISPR-Cas9-induced DSBs triggered ongoing chromosomal rearrangements, with 87% of structural variants not directly produced from the initial CRISPR-Cas9-induced DSBs, and chromosomal instability (CIN) peaking before cell death. By comparing the cytotoxicity of CRISPR-Cas9- to radiation-induced DSBs, we demonstrate that the number of DSBs required to achieve equitoxic effects was [~]3 times higher for radiation than CRISPR-Cas9. Finally, we show that PC cells that had survived CRISPR-Cas9 targeting retained susceptibility to subsequent CRISPR-Cas9-induced DSBs at different genomic sites with >87% growth inhibition. Together, our data support the therapeutic potential of CRISPR-Cas9 as an anti-cancer strategy.

cancer biology↗