Disruption of PH-kinase domain interactions leads to oncogenic activation of AKT in human cancers.

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.

Oncogenic NRAS, KRAS, and HRAS exhibit different leukemogenic potentials in mice.

RAS proteins are small GTPases that play a central role in transducing signals that regulate cell proliferation, survival, and differentiation. The RAS proteins interact with a common set of activators and effectors; however, they associate with different microdomains of the plasma membrane as well as other endomembranes and are capable of generating distinct signal outputs. Mutations that result in constitutive activation of RAS proteins are associated with approximately 30% of all human cancers; however, different RAS oncogenes are preferentially associated with different types of human cancer. In myeloid malignancies, NRAS mutations are more frequent than KRAS mutations, whereas HRAS mutations are rare. The mechanism underlying the different frequencies of RAS isoforms mutated in myeloid leukemia is not known. In this study, we compared the leukemogenic potential of activated NRAS, KRAS, and HRAS in the same bone marrow transduction/transplantation model system. We found that all three RAS oncogenes have the ability to induce myeloid leukemias, yet have distinct leukemogenic strengths and phenotypes. The models established here provide a system for further studying the molecular mechanisms in the pathogenesis of myeloid malignancies and for testing targeted therapies.

Mouse model for NRAS-induced leukemogenesis.

Mutations that result in constitutive activation of RAS proteins are common in human hematological malignancies. In addition, functional activation of the RAS pathway can occur in leukemias, either due to mutations in genes that code for proteins upstream of RAS or due to inactivation of negative regulators of RAS. However, despite this prominent association of RAS activation with human leukemias, its precise role in leukemogenesis is not known. Previous studies have met with limited success in developing relevant animal models for leukemogenesis by oncogenic NRAS, the most frequently mutated RAS gene in human leukemias, and have suggested that oncogenic RAS might only act as a secondary event in leukemogenesis. This chapter describes an efficient and relevant murine model for myeloid leukemias initiated by oncogenic NRAS using an improved bone marrow transduction/transplantation system. This model provides a system for further studying the molecular mechanisms in the pathogenesis of myeloid malignancies and for testing targeted therapies.

Oncogenic ERBB3 mutations in human cancers.

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.

Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer.

Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein-coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ∼27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.

Oncogenic NRAS rapidly and efficiently induces CMML- and AML-like diseases in mice.

Activating mutations in RAS, predominantly NRAS, are common in myeloid malignancies. Previous studies in animal models have shown that oncogenic NRAS is unable to induce myeloid malignancies effectively, and it was suggested that oncogenic NRAS might only act as a secondary mutation in leukemogenesis. In this study, we examined the leukemogenicity of NRAS using an improved mouse bone marrow transduction and transplantation model. We found that oncogenic NRAS rapidly and efficiently induced chronic myelomonocytic leukemia (CMML)- or acute myeloid leukemia (AML)- like disease in mice, indicating that mutated NRAS can function as an initiating oncogene in the induction of myeloid malignancies. In addition to CMML and AML, we found that NRAS induced mastocytosis in mice. This result indicates that activation of the RAS pathway also plays an important role in the pathogenesis of mastocytosis. The mouse model for NRAS leukemogenesis established here provides a system for further studying the molecular mechanisms in the pathogenesis of myeloid malignancies and for testing relevant therapies.