Kinase activation and transformation by NUP214-ABL1 is dependent on the context of the nuclear pore.

Genetic alterations causing constitutive tyrosine kinase activation are observed in a broad spectrum of cancers. Thus far, these mutant kinases have been localized to the plasma membrane or cytoplasm, where they engage proliferation and survival pathways. We report that the NUP214-ABL1 fusion is unique among these because of its requisite localization to the nuclear pore complex for its transforming potential. We show that NUP214-ABL1 displays attenuated transforming capacity as compared to BCR-ABL1 and that NUP214-ABL1 preferentially transforms T cells, which is in agreement with its unique occurrence in T cell acute lymphoblastic leukemia. Furthermore, NUP214-ABL1 differs from BCR-ABL1 in subcellular localization, initiation of kinase activity, and signaling and lacks phosphorylation on its activation loop. In addition to delineating an unusual mechanism for kinase activation, this study provides new insights into the spectrum of chromosomal translocations involving nucleoporins by indicating that the nuclear pore context itself may play a central role in transformation.

In vitro validation of gamma-secretase inhibitors alone or in combination with other anti-cancer drugs for the treatment of T-cell acute lymphoblastic leukemia.

BACKGROUND: Activating NOTCH1 mutations are common in T-cell acute lymphoblastic leukemia. Inhibition of NOTCH1 signaling with gamma-secretase inhibitors causes cell cycle block, but only after treatment for several days. We further documented the effects of gamma-secretase inhibitor treatment on T-cell acute lymphoblastic leukemia cell lines and tested whether combining gamma-secretase inhibitors with other anti-cancer drugs offers a therapeutic advantage. DESIGN AND METHODS: The effect of gamma-secretase inhibitor treatment and combinations of gamma-secretase inhibitors with chemotherapy or glucocorticoids was assessed on T-cell acute lymphoblastic leukemia cell lines. We sequenced NOTCH1 in T-cell acute lymphoblastic leukemia cases with ABL1 fusions and tested combinations of gamma-secretase inhibitors and the ABL1 inhibitor imatinib in a T-cell acute lymphoblastic leukemia cell line. RESULTS: gamma-secretase inhibitor treatment for 5-7 days reversibly inhibited cell proliferation, caused cell cycle block in sensitive T-cell acute lymphoblastic leukemia cell lines, and caused differentiation of some T-cell acute lymphoblastic leukemia cell lines. Treatment for 14 days or longer was required to induce significant apoptosis. The cytotoxic effects of the chemotherapeutic agent vincristine were not significantly enhanced by addition of gamma-secretase inhibitors to T-cell acute lymphoblastic leukemia cell lines, but gamma-secretase inhibitor treatment sensitized cells to the effect of dexamethasone. NOTCH1 mutations were identified in all T-cell acute lymphoblastic leukemia patients with ABL1 fusions and in a T-cell acute lymphoblastic leukemia cell line expressing NUP214-ABL1. In this cell line, the anti-proliferative effect of imatinib was increased by pre-treatment with gamma-secretase inhibitors. CONCLUSIONS: Short-term treatment of T-cell acute lymphoblastic leukemia cell lines with gamma-secretase inhibitors had limited effects on cell proliferation and survival. Combinations of gamma-secretase inhibitors with other drugs may be required to obtain efficient therapeutic effects in T-cell acute lymphoblastic leukemia, and not all combinations may be useful.

Efficacy of the kinase inhibitor SU11248 against gastrointestinal stromal tumor mutants refractory to imatinib mesylate.

PURPOSE: The majority of gastrointestinal stromal tumors harbor mutations in the receptor tyrosine kinases KIT or platelet-derived growth factor receptor A (PDGFRA), and respond to treatment with the tyrosine kinase inhibitor imatinib. Some tumors, however, show primary resistance to imatinib treatment, and most others become resistant during treatment. The most common mechanism of imatinib resistance involves specific mutations in the kinase domains of KIT or PDGFRA. We tested the activity of SU11248, an orally active small-molecule tyrosine kinase inhibitor, to inhibit important imatinib-resistant KIT and PDGFRA mutants. EXPERIMENTAL DESIGN: Primary imatinib-resistant tumor cells and cell lines expressing clinically identified imatinib-resistant KIT-V654A, KIT-T670I, or PDGFRA-D842V mutant isoforms were evaluated for sensitivity to SU11248 by Western immunoblotting and proliferation assays. Three patients with the KIT-V654A mutation were treated with SU11248. RESULTS: Based on ex vivo assays, SU11248 potently inhibits KIT kinase activity of V654A and T670I mutants and suppresses proliferation of the cells expressing these mutations. Sensitivity of KIT-V654A and KIT-T670I mutants to SU11248 was confirmed using cell lines expressing these mutants. In contrast, SU11248 did not potently inhibit the PDGFRA-D842V mutant. In agreement with these results, two of the three imatinib-resistant patients with the KIT-V654A mutation responded to SU11248 treatment. CONCLUSIONS: These studies suggest that SU11248 may be a useful therapeutic agent to treat gastrointestinal stromal tumors harboring the imatinib-resistant KIT-V654A or KIT-T670I mutations, but it has no effect on the activity of the PDGFRA-D842V mutant. Specific kinase inhibitors should be designed to inhibit the constitutive activating PDGFRA mutation at codon 842.

Sorafenib is a potent inhibitor of FIP1L1-PDGFRalpha and the imatinib-resistant FIP1L1-PDGFRalpha T674I mutant.

The FIP1L1-PDGFRA oncogene is a common cause of chronic eosinophilic leukemia (CEL), and encodes an activated tyrosine kinase that is inhibited by imatinib. FIP1L1-PDGFRA-positive patients with CEL respond to low-dose imatinib therapy, but resistance due to acquired T674I mutation has been observed. We report here the identification of sorafenib as a potent inhibitor of the FIP1 like 1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRalpha) (T674I) mutant. Sorafenib inhibited the proliferation of FIP1L1-PDGFRalpha and FIP1L1-PDGFRalpha(T674I)-transformed Ba/F3 cells and induced apoptosis of the EOL-1 cell line at a low nanomolar concentration. Western blot analysis confirmed that these effects were due to a direct effect on FIP1L1-PDGFRalpha and FIP1L1-PDGFRalpha(T674I). Sorafenib was recently approved for the treatment of renal cell carcinoma. Our data suggest that low doses of sorafenib could be efficient for the treatment of FIP1L1-PDGFRA-positive CEL and could be used to overcome resistance to imatinib associated with the T674I mutation.

Activation of FIP1L1-PDGFRalpha requires disruption of the juxtamembrane domain of PDGFRalpha and is FIP1L1-independent.

Genetic abnormalities that result in expression of chimeric tyrosine kinase proteins such as BCR-ABL1 and ETV6-PDGFRbeta are common causes of hematopoietic malignancies. The paradigm for constitutive activation of these fusion tyrosine kinases is enforced homodimerization by self-association domains present in the fusion partner proteins. The unique interstitial deletion on chromosome 4q12 that leads to expression of the FIP1L1-PDGFRalpha fusion tyrosine kinase was recently identified as a cause of chronic eosinophilic leukemia. In this report, we demonstrate that FIP1L1 is completely dispensable for PDGFRalpha activation in vitro and in vivo. Instead, truncation of PDGFRalpha between two conserved tryptophan residues in the juxtamembrane (JM) domain is required for kinase activation and transforming potential of FIP1L1-PDGFRalpha. The presence of a complete JM domain in FIP1L1-PDGFRalpha is inhibitory, but this autoinhibition can be overcome by enforced homodimerization. Similar effects of the JM domain in the context of PDGFRbeta were observed. These results suggest that disruption of the autoinhibitory JM domain is an alternative, dimerization-independent mechanism by which chimeric tyrosine kinases are constitutively activated and induce leukemogenesis.