Efficient Correction of Oncogenic KRAS and TP53 Mutations through CRISPR Base Editing.

KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. Here, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS. However, analysis of the remaining cell population after long-term Cas9 expression unmasked the occurence of oncogenic KRAS escape variants that were resistant to Cas9-cleavage. In contrast, the use of an adenine base editor to correct oncogenic KRAS mutations progressively depleted the targeted cells without the appearance of escape variants and allowed efficient and simultaneous correction of a cancer-associated TP53 mutation. Oncogenic KRAS and TP53 base editing was possible in patient-derived cancer organoids, suggesting that base editor approaches to correct oncogenic mutations could be developed for functional interrogation of vulnerabilities in a personalized manner for future precision oncology applications. SIGNIFICANCE: Repairing KRAS mutations with base editors can be used for providing a better understanding of RAS biology and may lay the foundation for improved treatments for KRAS-mutant cancers.

Using CRISPR-Cas9 to Dissect Cancer Mutations in Cell Lines.

The CRISPR-Cas9 technology has revolutionized the scope and pace of biomedical research, enabling the targeting of specific genomic sequences for a wide spectrum of applications. Here we describe assays to functionally interrogate mutations identified in cancer cells utilizing both CRISPR-Cas9 nuclease and base editors. We provide guidelines to interrogate known cancer driver mutations or functionally screen for novel vulnerability mutations with these systems in characterized human cancer cell lines. The proposed platform should be transferable to primary cancer cells, opening up a path for precision oncology on a functional level.

RNAi-Mediated Screen of Primary AML Cells Nominates MDM4 as a Therapeutic Target in NK-AML with DNMT3A Mutations.

DNA-methyltransferase 3A (DNMT3A) mutations belong to the most frequent genetic aberrations found in adult acute myeloid leukemia (AML). Recent evidence suggests that these mutations arise early in leukemogenesis, marking leukemic progenitors and stem cells, and persist through consolidation chemotherapy, providing a pool for AML relapse. Currently, there are no therapeutic approaches directed specifically against this cell population. To unravel therapeutically actionable targets in mutant DNMT3A-driven AML cells, we have performed a focused RNAi screen in a panel of 30 primary AML samples, all carrying a DNMT3A R882 mutation. As one of the strongest hits, we identified MDM4 as a gene essential for proliferation of primary DNMT3AWT/R882X AML cells. We analyzed a publicly available RNA-Seq dataset of primary normal karyotype (NK) AML samples and found a trend towards MDM4 transcript overexpression particularly in DNMT3A-mutant samples. Moreover, we found that the MDM2/4 inhibitor ALRN-6924 impairs growth of DNMT3AWT/R882X primary cells in vitro by inducing cell cycle arrest through upregulation of p53 target genes. Our results suggest that MDM4 inhibition is a potential target in NK-AML patients bearing DNMT3A R882X mutations.

CRISPR/Cas9 as a tool to dissect cancer mutations.

The CRISPR/Cas9 system is transforming many biomedical disciplines, including cancer research. Through its flexible programmability and efficiency to induce DNA double strand breaks it has become straightforward to introduce cancer mutations into cells in vitro and/or in vivo. However, not all mutations contribute equally to tumorigenesis and distinguishing essential mutations for tumor growth and survival from biologically inert mutations is cumbersome. Here we present a method to screen for the functional relevance of mutations in high throughput in established cancer cell lines. We employ the CRISPR/Cas9 system to probe cancer vulnerabilities in a colorectal carcinoma cell line in an attempt to identify novel cancer driver mutations. We designed 100 high quality sgRNAs that are able to specifically cleave mutations present in the colorectal carcinoma cell line RKO. An all-in-one lentiviral library harboring these sgRNAs was then generated and used in a pooled screen to probe possible growth dependencies on these mutations. Genomic DNA at different time points were collected, the sgRNA cassettes were PCR amplified, purified and sgRNA counts were quantified by means of deep sequencing. The analysis revealed two sgRNAs targeting the same mutation (UTP14A: S99delS) to be depleted over time in RKO cells. Validation and characterization confirmed that the inactivation of this mutation impairs cell growth, nominating UTP14A: S99delS as a putative driver mutation in RKO cells. Overall, our approach demonstrates that the CRISPR/Cas9 system is a powerful tool to functionally dissect cancer mutations at large-scale.

Reoperation for left atrioventricular valve failure in repaired atrioventricular septal defect: Can more valves be preserved in the current era?

OBJECTIVE: Left atrio-ventricular valve (LAVV) regurgitation after repair of an atrio-ventricular septal defect (AVSD) may necessitate further surgery. However, redo-LAVV repair remains challenging. We sought to determine if more LAVV valves are preserved in the current era, and analyze early and longer-term results. PATIENTS: All consecutive patients with repaired AVSD who underwent redo-LAVV surgery from January 2004 to April 2017 were included. Patients with single ventricles, atrial isomerism, and complex associated anomalies were excluded. METHODS: This was a single-center study using retrospective chart review and an institutional database for follow-up information. Data analyzed included number and year of primary AVSD and redo-LAVV operation, presence of trisomy 21, morphology of AVSD, mortality, and reoperation. Univariate analysis included repair and replacement rates and early and long-term survival. RESULTS: During the study period 36 redo-LAVV operations were performed, with repair in 28 and replacement in eight. The number of redo-operations increased from 13 in the first part to 23 in the second part of the study. The rate of LAVV preservation significantly increased over time (54% vs 91%, P < 0.01), and was not affected by morphology of AVSD or trisomy 21. There was one in-hospital death at Day 42 and overall estimated survival was 94.5% at 5 years. Freedom from reoperation after redo-LAVV repair was 87% at 5 years with no significant difference between repair and replacement groups. CONCLUSION: In the current era, more LAVVs can be preserved at the time of redo-operation with excellent early and long-term survival and acceptable reoperation rates. LAVV morphology and presence of trisomy 21 did not affect outcome.