CRISPR/Cas9 induces DNA double-strand breaks that are repaired by cell-autonomous repair pathways, namely, non-homologous end-joining (NHEJ), or homology-directed repair (HDR). While HDR is absent in ...G1, NHEJ is active throughout the cell cycle and, thus, is largely favored over HDR. We devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell-cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1, resulting in a cell-cycle-tailored expression with low levels in G1 but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the rate of HDR by up to 87% compared to wild-type Cas9. Future developments may enable high-resolution expression of genome engineering proteins, which might increase HDR rates further, and may contribute to a better understanding of DNA repair pathways due to spatiotemporal control of DNA damage induction.
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•Homology-directed repair (HDR) enables generation of site-specific Knockins•HDR is restricted to S/G2 and competes with non-homologous end-joining repair•Cas9-hGem(1/110), a cell-cycle-tailored genome-editing tool, is generated•Cas9-hGem(1/110) increases the rate of HDR up to 1.87-fold compared to wild-type Cas9
Using a protein engineering approach, Gutschner et al. generate a Cas9 fusion protein to control genome editing in time and space. Coupling Cas9 protein levels to cell-cycle dynamics results in higher site-specific integration events.
The genome of pancreatic ductal adenocarcinoma (PDAC) frequently contains deletions of tumour suppressor gene loci, most notably SMAD4, which is homozygously deleted in nearly one-third of cases. As ...loss of neighbouring housekeeping genes can confer collateral lethality, we sought to determine whether loss of the metabolic gene malic enzyme 2 (ME2) in the SMAD4 locus would create cancer-specific metabolic vulnerability upon targeting of its paralogous isoform ME3. The mitochondrial malic enzymes (ME2 and ME3) are oxidative decarboxylases that catalyse the conversion of malate to pyruvate and are essential for NADPH regeneration and reactive oxygen species homeostasis. Here we show that ME3 depletion selectively kills ME2-null PDAC cells in a manner consistent with an essential function for ME3 in ME2-null cancer cells. Mechanistically, integrated metabolomic and molecular investigation of cells deficient in mitochondrial malic enzymes revealed diminished NADPH production and consequent high levels of reactive oxygen species. These changes activate AMP activated protein kinase (AMPK), which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 branched-chain amino acid transaminase 2) gene. BCAT2 catalyses the transfer of the amino group from branched-chain amino acids to α-ketoglutarate (α-KG) thereby regenerating glutamate, which functions in part to support de novo nucleotide synthesis. Thus, mitochondrial malic enzyme deficiency, which results in impaired NADPH production, provides a prime 'collateral lethality' therapeutic strategy for the treatment of a substantial fraction of patients diagnosed with this intractable disease.
A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here, we explored the role of oncogenic KRAS in ...protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS*) drives cell-autonomous expression of type I cytokine receptor complexes (IL2rγ-IL4rα and IL2rγ-IL13rα1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS* and Th2 cells producing IL4 and IL13. Activated IL2rγ-IL4rα and IL2rγ-IL13rα1 receptors signal primarily via JAK1-STAT6. Integrated transcriptomic, chromatin occupancy, and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus, paracrine signaling in the tumor microenvironment plays a key role in the KRAS*-driven metabolic reprogramming of PDAC. SIGNIFICANCE: Type II cytokines, secreted by Th2 cells in the tumor microenvironment, can stimulate cancer cell-intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS*-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions, providing candidate therapeutic targets in the KRAS* pathway for this intractable disease.
Pancreatic ductal adenocarcinoma (PDAC) is still one of the deadliest cancers in oncology because of its increasing incidence and poor survival rate. More than 90% of PDAC patients are KRAS mutated ...(KRASmu), with KRASG12D and KRASG12V being the most common mutations. Despite this critical role, its characteristics have made direct targeting of the RAS protein extremely difficult. KRAS regulates development, cell growth, epigenetically dysregulated differentiation, and survival in PDAC through activation of key downstream pathways, such as MAPK-ERK and PI3K-AKT-mammalian target of rapamycin (mTOR) signaling, in a KRAS-dependent manner. KRASmu induces the occurrence of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) and leads to an immunosuppressive tumor microenvironment (TME). In this context, the oncogenic mutation of KRAS induces an epigenetic program that leads to the initiation of PDAC. Several studies have identified multiple direct and indirect inhibitors of KRAS signaling. Therefore, KRAS dependency is so essential in KRASmu PDAC that cancer cells have secured several compensatory escape mechanisms to counteract the efficacy of KRAS inhibitors, such as activation of MEK/ERK signaling or YAP1 upregulation. This review will provide insights into KRAS dependency in PDAC and analyze recent data on inhibitors of KRAS signaling, focusing on how cancer cells establish compensatory escape mechanisms.
Following mutations in BRAF and NRAS, the RAC1 c.85C>T single-nucleotide variant (SNV) encoding P29S amino acid change represents the next most frequently observed protein-coding hotspot mutation in ...melanoma. However, the biologic and clinical significance of the RAC1 P29S somatic mutation in approximately 4% to 9% of patients remains unclear. Here, we demonstrate that melanoma cell lines possessing the RAC1 hotspot variant are resistant to RAF inhibitors (vemurafenib and dabrafenib). Enforced expression of RAC1 P29S in sensitive BRAF-mutant melanoma cell lines confers resistance manifested by increased viability, decreased apoptosis, and enhanced tumor growth in vivo upon treatment with RAF inhibitors. Conversely, RNAi-mediated silencing of endogenous RAC1 P29S in a melanoma cell line with a co-occurring BRAF V600 mutation increased sensitivity to vemurafenib and dabrafenib. Our results suggest RAC1 P29S status may offer a predictive biomarker for RAF inhibitor resistance in melanoma patients, where it should be evaluated clinically.
Cellular dedifferentiation is a key mechanism driving cancer progression. Acquisition of mesenchymal features has been associated with drug resistance, poor prognosis, and disease relapse in many ...tumor types. Therefore, successful targeting of tumors harboring these characteristics is a priority in oncology practice. The SWItch/Sucrose non-fermentable (SWI/SNF) chromatin remodeling complex has also emerged as a critical player in tumor progression, leading to the identification of several SWI/SNF complex genes as potential disease biomarkers and targets of anticancer therapies. AT-rich interaction domain-containing protein 1A (ARID1A) is a component of SWI/SNF, and mutations in
represent one of the most frequent molecular alterations in human cancers.
mutations occur in approximately 10% of pancreatic ductal adenocarcinomas (PDAC), but whether these mutations confer a therapeutic opportunity remains unclear. Here, we demonstrate that loss of ARID1A promotes an epithelial-mesenchymal transition (EMT) phenotype and sensitizes PDAC cells to a clinical inhibitor of HSP90, NVP-AUY922, both
and
. Although loss of ARID1A alone did not significantly affect proliferative potential or rate of apoptosis, ARID1A-deficient cells were sensitized to HSP90 inhibition, potentially by promoting the degradation of intermediate filaments driving EMT, resulting in cell death. Our results describe a mechanistic link between ARID1A defects and a quasi-mesenchymal phenotype, suggesting that deleterious mutations in
associated with protein loss exhibit potential as a biomarker for patients with PDAC who may benefit by HSP90-targeting drugs treatment. SIGNIFICANCE: This study identifies ARID1A loss as a promising biomarker for the identification of PDAC tumors that are potentially responsive to treatment with proteotoxic agents.
Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Here, studying clonal evolution of tumor cells derived from ...human pancreatic tumors, we demonstrate that in vitro cultures and in vivo tumors are maintained by a common set of tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of adaptive responses to therapeutics, we uncovered a multitude of functionally heterogeneous subpopulations of cells with differential degrees of drug sensitivity. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying functionally diverse subpopulations. Molecular annotation of gemcitabine-naive clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy, representing a potential biomarker to stratify patients with pancreatic cancer.
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•Lineage tracing reveals in vitro and in vivo tumors are maintained by common clones•Deep lineage dynamics characterization enables generation of clonal replica tumors•Pancreatic CRTs unmask functional tumor heterogeneity in response to therapeutics•Gemcitabine-naive subclonal gene signature predicts chemotherapy response
High-complexity lineage tracing shows that tumors growing in different environments are maintained by a common set of tumorigenic cells that enables the generation of clonal replica tumors (CRTs). Applying CRTs, Seth et al. unmask functional heterogeneity in response to therapeutics and identify a signature that predicts chemoresistance in pancreatic cancer.
Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial ...metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.
We present, to our knowledge, the first reported case of germline neurofibromatosis Type 2 (NF2) associated with renal cell carcinoma unclassified with medullary phenotype (RCCU-MP) with somatic loss ...by immunohistochemistry of the
tumor suppressor gene located centromeric to NF2 on chromosome 22q. Our patient is a 15-year-old with germline neurofibromatosis Type 2 (NF2) confirmed by pathogenic mutation of c.-854-??46+??deletion. Her NF2 history is positive for a right optic nerve sheath meningioma, CNIII schwannoma requiring radiation therapy and post gross total resection of right frontotemporal anaplastic meningioma followed by radiation. At age 15 she developed new onset weight loss and abdominal pain due to RCCU-MP. Hemoglobin electrophoresis was negative for sickle hemoglobinopathy. Chemotherapy (cisplatin, gemcitabine and paclitaxel) was initiated followed by radical resection. Given the unique renal pathology of a high grade malignancy with loss of SMARCB1 expression via immunohistochemistry, and history of meningioma with MLH1 loss of expression and retained expression of PMS2, MSH2 and MSH6, further germline genetic testing was sent for
and mismatch repair syndromes. Germline testing was negative for mutation in
. Therefore, this is the first reported case of RCCU-MP associated with germline
mutation. This suggests the importance of closer surveillance in the adolescent and young adult population with NF2 with any suspicious findings of malignancy outside of the usual scope of practice with NF2.
•The therapeutic landscape for metastatic renal cell carcinoma is rapidly evolving.•Second-Generation p-values can facilitate interpretation of trial evidence.•We reviewed all pivotal therapeutic ...trials for metastatic renal cell carcinoma.•We provide a treatment algorithm reflecting the conclusions of our review.
The therapeutic options for advanced or metastatic renal cell carcinoma (mRCC) have drastically evolved over the past 20 years. High-dose interleukin-2 (HD IL-2), which led to durable complete responses in a small fraction of patients by activating the interleukin-2 (IL-2) pathway, faded in popularity with the advent of oral tyrosine kinase inhibitors directed against the VEGF pathway (VEGFR-TKI) showing better tolerability, wider applicability, higher objective response rates, and longer progression-free survival than HD IL-2. More recently, new insights on how to more efficiently harness the immune system led to the development of immune checkpoint inhibitor (ICI) therapies, which rapidly became an integral component of mRCC treatment. The recently approved regimen combining the PD-1 inhibitor, nivolumab, and the CTLA-4 inhibitor, ipilimumab, and the recently approved regimens combining the oral VEGFR-TKI, axitinib, with the PD-1 inhibitor, pembrolizumab, or the PD-L1 inhibitor, avelumab, were shown to yield improved outcomes compared with sunitinib, the VEGFR-TKI that was used as a comparator. The present review discusses the evidence behind the treatment approvals for mRCC and provides an overview of the current therapeutic landscape. We evaluated the results of randomized clinical trials for mRCC based on the effect size differences between treatments on relative scales and used second-generation p-values as a descriptive summary of the statistical evidence.
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