Genome-wide CRISPR screens identify PKMYT1 as a therapeutic target in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with an overall 5-year survival rate of <12% due to the lack of effective treatments. Novel treatment strategies are urgently needed. Here, PKMYT1 is identified through genome-wide CRISPR screens as a non-mutant, genetic vulnerability of PDAC. Higher PKMYT1 expression levels indicate poor prognosis in PDAC patients. PKMYT1 ablation inhibits tumor growth and proliferation in vitro and in vivo by regulating cell cycle progression and inducing apoptosis. Moreover, pharmacological inhibition of PKMYT1 shows efficacy in multiple PDAC cell models and effectively induces tumor regression without overt toxicity in PDAC cell line-derived xenograft and in more clinically relevant patient-derived xenograft models. Mechanistically, in addition to its canonical function of phosphorylating CDK1, PKMYT1 functions as an oncogene to promote PDAC tumorigenesis by regulating PLK1 expression and phosphorylation. Finally, TP53 function and PRKDC activation are shown to modulate the sensitivity to PKMYT1 inhibition. These results define PKMYT1 dependency in PDAC and identify potential therapeutic strategies for clinical translation.

E3 ligase MKRN3 is a tumor suppressor regulating PABPC1 ubiquitination in non-small cell lung cancer.

Central precocious puberty (CPP), largely caused by germline mutations in the MKRN3 gene, has been epidemiologically linked to cancers. MKRN3 is frequently mutated in non-small cell lung cancers (NSCLCs) with five cohorts. Genomic MKRN3 aberrations are significantly enriched in NSCLC samples harboring oncogenic KRAS mutations. Low MKRN3 expression levels correlate with poor patient survival. Reconstitution of MKRN3 in MKRN3-inactivated NSCLC cells directly abrogates in vitro and in vivo tumor growth and proliferation. MKRN3 knockout mice are susceptible to urethane-induced lung cancer, and lung cell-specific knockout of endogenous MKRN3 accelerates NSCLC tumorigenesis in vivo. A mass spectrometry-based proteomics screen identified PABPC1 as a major substrate for MKRN3. The tumor suppressor function of MKRN3 is dependent on its E3 ligase activity, and MKRN3 missense mutations identified in patients substantially compromise MKRN3-mediated PABPC1 ubiquitination. Furthermore, MKRN3 modulates cell proliferation through PABPC1 nonproteolytic ubiquitination and subsequently, PABPC1-mediated global protein synthesis. Our integrated approaches demonstrate that the CPP-associated gene MKRN3 is a tumor suppressor.

Integrated Screens Identify CDK1 as a Therapeutic Target in Advanced Gastrointestinal Stromal Tumors.

Oncogenic KIT or PDGFRA receptor tyrosine kinase mutations are compelling therapeutic targets in gastrointestinal stromal tumor (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with advanced GIST. Polyclonal emergence of KIT/PDGFRA secondary mutations is the main mechanism of imatinib progression, making it challenging to overcome KIT/PDGFRA-inhibitor resistance. It is unclear whether there are other therapeutic targets in advanced GIST. Using genome-wide transcriptomic profiling of advanced versus early-stage GIST and CRISPR knockout functional screens, we demonstrate that CDK1 is frequently highly expressed in advanced GIST but not in early-stage GIST across three patient cohorts. High expression of CDK1 was associated with malignancy in GIST. CDK1 was critically required for advanced GIST, including imatinib-resistant GIST. CDK1 ablation led to robust proliferation inhibition. A mass spectrometry-based proteomics screen further revealed that AKT is a novel substrate of CDK1 kinase in GIST. CDK1 bound AKT and regulated its phosphorylation, thereby promoting GIST proliferation and progression. Importantly, a pharmacologic inhibitor of CDK1, RO-3306, disrupted GIST cell proliferation in CDK1 highly expressed GIST but not in CDK1-negative GIST cells and nontransformed fibroblast cells. Treatment with RO-3306 reduced tumor growth in both imatinib-resistant and imatinib-sensitive GIST xenograft mouse models. Our findings suggest that CDK1 represents a druggable therapeutic target in GIST and warrants further testing in clinical trials. SIGNIFICANCE: These findings propose CDK1 as a novel cell-cycle-independent vulnerability in gastrointestinal stromal tumors, representing a new therapeutic opportunity for patients with advanced disease.

Oncogenic and drug-sensitive RET mutations in human epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is a highly lethal malignancy. Improvement in genetic characterization of EOC patients is required to propose new potential targets, since surgical resection coupled to chemotherapy, presents several limits such as cancer recurrence and drug resistance. Targeted therapies have more efficacy and less toxicity than standard treatments. One of the most relevant cancer-specific actionable targets are protein tyrosine kinases (PTKs) whose role in EOC need to be better investigated. METHODS: EOC genomic datasets are retrieved and analyzed. The biological and clinical significance of RET genomic aberrations in ovarian cancer context are investigated by a series of in vitro and in vivo experiments. RESULTS: Epithelial ovarian cancer sequencing projects identify recurrent genomic RET missense mutations in 1.98% of patients, ranking as the top-five hit among the 100 receptor tyrosine kinases-encoding genes. RET mutants R693H and A750T show oncogenic transformation properties in NIH3T3 cells. Introduction of the RET mutants into human EOC cells increases RET signaling, cell viability, anchorage-independent cell growth and tumor xenograft growth in nude mice, demonstrating that they are activating mutations. RET mutants significantly enhance the activation of RET and its downstream MAPK and AKT signaling pathway in ovarian cancer cells. Vandetanib, a clinical approved RET inhibitor, inhibits the cell viability and decreases the activation of RET-MAPK signaling pathways in EOC cells expressing oncogenic RET mutants. CONCLUSIONS: The discovery of RET pathogenic variants in the EOC patients, suggests a previously underestimated role for RET in EOC tumorigenesis. The identification of the gain-of-function RET mutations in EOC highlights the potential use of RET in targeted therapy to treat ovarian cancer patients.

Spectrum of activity of dasatinib against mutant KIT kinases associated with drug-sensitive and drug-resistant gastrointestinal stromal tumors.

BACKGROUND: The majority of GISTs express mutationally activated KIT. Imatinib and sunitinib are approved KIT-inhibiting therapies. Their efficacy is usually hampered by the acquired multiple secondary drug-resistance KIT mutations. The most problematic resistance subset is GISTs with acquisition of secondary mutations in the KIT activation loop. Here, we establish the spectrum of activity of dasatinib against a comprehensive collection of clinically relevant KIT mutants associated with drug-sensitive and drug-resistant GIST. METHODS: The cellular and in vitro activities of tyrosine kinase inhibitors (TKIs) against mutant KIT were assessed using a panel of engineered and GIST-derived cell lines. The in vivo activities of dasatinib were determined using TKI-resistant xenograft models. RESULTS: In engineered and GIST-derived cell lines, dasatinib potently inhibited KIT with primary mutations in exon 11 or 9 and a range of secondary imatinib-resistant mutations in exons 13 and 14, encoding the ATP-binding pocket, and in exons 17 and 18, encoding the activation loop, with the exception of a substitution at codon T670. Our data show that dasatinib is more potent than imatinib or sunitinib at inhibiting the activity of drug-resistant KIT mutants. Dasatinib also induces regression in GIST-derived xenograft models containing these secondary mutations. A major determinant of the efficacy of dasatinib for the treatment of advanced GIST is the activity of this inhibitor against KIT mutants. CONCLUSION: Dasatinib shows efficacy in cancer models, inhibiting a wide range of oncogenic primary and drug-resistant KIT mutants. These results have implications for the further development of dasatinib precision therapy in GIST patients.

Oncogenic ERBB2 aberrations and KRAS mutations cooperate to promote pancreatic ductal adenocarcinoma progression.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with few therapeutic options, representing one of the great challenges in oncology. Activating KRAS mutation, occurring in >90% PDACs, is present in pancreatic intraepithelial neoplasia lesions, the precursor ductal lesions of PDAC, indicating additional genetic alterations contribute to the pathogenesis of PDAC. PDAC sequencing projects identify recurrent genomic ERBB2 alterations, mutations and amplifications, in 8.5% of PDAC patients, ranking as the top hit among the 100 receptor tyrosine kinases-encoding genes. Introduction of the ERBB2 mutations encoding protein variants S310F, S423R, R678Q, Q679L, E717D, L755S, V777L and V842I into human pancreatic epithelial cells causes oncogenic transformation, increasing ERBB2 signaling, anchorage-independent cell growth and tumor xenograft growth in nude mice, demonstrating that they are activating mutations. Interestingly, in many PDACs, mutations in ERBB2 and KRAS occur together. ERBB2 activating mutants facilitate KRAS-driven oncogenic properties. Introduction of ERBB2 mutations into KRAS-mutant PDAC cells activates ERBB2 signaling, promotes tumor growth and attenuates KRAS dependency. In contrast, a CRISPR-mediated knockout (KO) of ERBB2 in ERBB2-amplified PDAC cells inhibits ERBB2 signaling, colony formation, anchorage-independent growth and tumor xenograft formation. Finally, oncogenic ERBB2 aberrations can be abrogated by treatment with small-molecule inhibitors. ERBB2 and KRAS inhibition cooperate to suppress PDAC cell growth in vitro and to promote tumor regression in nude mice, providing a rationale for testing an anti-ERBB2 drug in combination with a KRAS inhibitor in ERBB2-mutant PDAC patients that are currently untreatable.

Mutational inactivation of mTORC1 repressor gene DEPDC5 in human gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common human sarcoma and are initiated by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. Chromosome 22q deletions are well-recognized frequent abnormalities in GISTs, occurring in ∼50% of GISTs. These deletions are thought to contribute to the pathogenesis of this disease via currently unidentified tumor suppressor mechanisms. Using whole exome sequencing, we report recurrent genomic inactivated DEPDC5 gene mutations in GISTs (16.4%, 9 of 55 patients). The demonstration of clonal DEPDC5 inactivation mutations in longitudinal specimens and in multiple metastases from individual patients suggests that these mutations have tumorigenic roles in GIST progression. DEPDC5 inactivation promotes GIST tumor growth in vitro and in nude mice. DEPDC5 reduces cell proliferation through the mTORC1-signaling pathway and subsequently induces cell-cycle arrest. Furthermore, DEPDC5 modulates the sensitivity of GIST to KIT inhibitors, and the combination therapy with mTOR inhibitor and KIT inhibitor may work better in GIST patients with DEPDC5 inactivation. These findings of recurrent genomic alterations, together with functional data, validate the DEPDC5 as a bona fide tumor suppressor contributing to GIST progression and a biologically relevant target of the frequent chromosome 22q deletions.

Genetic progression in gastrointestinal stromal tumors: mechanisms and molecular interventions.

Gastrointestinal stromal tumors (GISTs) are the most common sarcomas in humans. Constitutively activating mutations in the KIT or PDGFRA receptor tyrosine kinases are the initiating oncogenic events. Most metastatic GISTs respond dramatically to therapies with KIT/PDGFRA inhibitors. Asymptomatic and mitotically-inactive KIT/PDGFRA-mutant "microGISTs" are found in one third of adults, but most of these small tumors never progress to malignancy, underscoring that a progression of oncogenic mutations is required. Recent studies have identified key genomic abnormalities in GIST progression. Novel insights into the genetic progression of GISTs are shedding new light on therapeutic innovations.