CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within noncoding regions. We have adapted the CRISPR-Cas9 gene editing tool as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks. Whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002) showed an average of 417 somatic PAMs per tumor produced from single base substitutions. Further analyses of 591 paired T-N samples from The International Cancer Genome Consortium found medians of ∼455 somatic PAMs per tumor in pancreatic, ∼2800 in lung, and ∼3200 in esophageal cancer cohorts. Finally, we demonstrated 69-99% selective cell death of three targeted pancreatic cancer cell lines using 4-9 sgRNAs designed using the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs in either the patient's normal cells or an irrelevant cancer using WGS. This study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy, and supports the genetic targeting of adult cancers.

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

Introduction: Metastatic 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. Methods: Whole 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. Results: We 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. Conclusions: Regions 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.

Tumor- and Nerve-Derived Axon Guidance Molecule Promotes Pancreatic Ductal Adenocarcinoma Progression and Metastasis through Macrophage Reprogramming.

Axon guidance molecules were found to be the gene family most frequently altered in pancreatic ductal adenocarcinoma (PDA) through mutations and copy number changes. However, the exact molecular mechanism regarding PDA development remained unclear. Using genetically engineered mouse models to examine one of the axon guidance molecules, semaphorin 3D (SEMA3D), we found a dual role for tumor-derived SEMA3D in malignant transformation of pancreatic epithelial cells and a role for nerve-derived SEMA3D in PDA development. This was demonstrated by the pancreatic-specific knockout of the SEMA3D gene from the KRAS G12D and TP53 R 172 H mutation knock-in, PDX1-Cre (KPC) mouse model which demonstrated a delayed tumor initiation and growth comparing to the original KPC mouse model. Our results showed that SEMA3D knockout skews the macrophages in the pancreas away from M2 polarization, providing a potential mechanistic role of tumor-derived SEMA3D in PDA development. The KPC mice with the SEMA3D knockout remained metastasis-free, however, died from primary tumor growth. We then tested the hypothesis that a potential compensation mechanism could result from SEMA3D which is naturally expressed by the intratumoral nerves. Our study further revealed that nerve-derived SEMA3D does not reprogram macrophages directly, but reprograms macrophages indirectly through ARF6 signaling and lactate production in PDA tumor cells. SEMA3D increases tumor-secreted lactate which is sensed by GPCR132 on macrophages and subsequently stimulates pro-tumorigenic M2 polarization in vivo. Tumor intrinsic- and extrinsic-SEMA3D induced ARF6 signaling through its receptor Plexin D1 in a mutant KRAS-dependent manner. Consistently, RNA sequencing database analysis revealed an association of higher KRAS MUT expression with an increase in SEMA3D and ARF6 expression in human PDAs. Moreover, multiplex immunohistochemistry analysis showed an increased number of M2-polarized macrophages proximal to nerves in human PDA tissue expressing SEMA3D. Thus, this study suggests altered expression of SEMA3D in tumor cells lead to acquisition of cancer-promoting functions and the axon guidance signaling originating from nerves is "hijacked" by tumor cells to support their growth. Other axon guidance and neuronal development molecules may play a similar dual role which is worth further investigation. One sentence summary: Tumor- and nerve-derived SEMA3D promotes tumor progression and metastasis through macrophage reprogramming in the tumor microenvironment. STATEMENT OF SIGNIFICANCE: This study established the dual role of axon guidance molecule, SEMA3D, in the malignant transformation of pancreatic epithelial cells and of nerve-derived SEMA3D in PDA progression and metastasis. It revealed macrophage reprogramming as the mechanism underlying bothroles. Together, this research elucidated how inflammatory responses promote invasive PDA progression and metastasis through an oncogenic process.

CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic 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.

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

When we transduced pancreatic cancers with sgRNAs that targeted 2-16 target sites in the human genome, we found that increasing the number of CRISPR-Cas9 target sites produced greater cytotoxicity, with >99% growth inhibition observed by targeting only 12 sites. However, cell death was delayed by 2-3 weeks after sgRNA transduction, in contrast to the repair of double strand DNA breaks (DSBs) that happened within 3 days after transduction. To explain this discrepancy, we used both cytogenetics and whole genome sequencing to interrogate the genome. We first detected chromatid and chromosome breaks, followed by radial formations, dicentric, ring chromosomes, and other chromosomal aberrations that peaked at 14 days after transduction. Structural variants (SVs) were detected at sites that were directly targeted by CRISPR-Cas9, including SVs generated from two sites that were targeted, but the vast majority of SVs (89.4%) were detected elsewhere in the genome that arose later than those directly targeted. Cells also underwent polyploidization that peaked at day 10 as detected by XY FISH assay, and ultimately died via apoptosis. Overall, we found that the simultaneous DSBs induced by CRISPR-Cas9 in pancreatic cancers caused chromosomal instability and polyploidization that ultimately led to delayed cell death.

Menin regulates the serine biosynthetic pathway in Ewing sarcoma.

Developmental transcription programs are epigenetically regulated by multi-protein complexes, including the menin- and MLL-containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the menin-MLL interaction leads to loss of menin and MLL1 protein expression, and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of menin-MLL-mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bru-seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the menin-MLL interaction inhibitor MI-503. Menin-MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MI-503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability, and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with 13 C-labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by menin-dependent epigenetic mechanisms involving trithorax complexes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.