NOTCH mutations leading to increased receptor signaling

Abstract

The present invention is based upon the identification of regions within the NOTCH-1 receptor that, when mutated, lead to increased receptor signaling. The mutations are associated with uncontrolled cellular growth and this growth may be arrested using agents that interfere with NOTCH-1 activity, such as inhibitors of gamma-secretase. Assays for the NOTCH-1 mutations may be used diagnostically or as part of a treatment regimen for cancer patients.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to, and the benefit of, U.S. provisional applications 60 / 598,546, filed on Aug. 4, 2004 and 60 / 672,053, filed on Apr. 18, 2005. The contents of these prior applications are hereby incorporated by reference in their entirety.STATEMENT OF GOVERNMENT FUNDING [0002] The United States Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others under reasonable terms as provided for by the terms of NIH Grant No. R01CA082308, R01CA92433 and P01CA68484 awarded by the Department of Health and Human Services.FIELD OF THE INVENTION [0003] The present invention is directed to human NOTCH receptors that have undergone mutations resulting in either increased responsiveness to biological stimuli or constitutive activation independent of the need for other stimuli. The mutations occur in specific, well-defined, regions of NOTCH receptors ...

AI Technical Summary

Benefits of technology

[0010] The present invention is based upon the discovery that mutations in two specific regions of the NOTCH-1 receptor lead to increased receptor signaling and are associated with uncontrolled cellular growth. The first is a region within the heterodimerization domain that is characterized by the presence of many amino acids that are highly conserved between species. The amino acids (residues 1571-1622 in SEQ ID NO:1, see FIGS. 1 and 3 for sequence identification numbers) are encoded by exon 26 and are found within the NEC subunit (i.e. the extracellular portion) of the NOTCH-1 protein. In intact NOTCH-1, they are non-covalently bound to amino acids encoded by exon 27 (amino acids 1674-1730) which lie on the NTM subunit (i.e., the transmembrane portion of the receptor). It is believed that mutations, especially non-conservative mutations, in these “hotspots” of the HD domain, i.e., 1571-1622 of the NEC subunit and 1674-1730 of the NTM subunit, destabilize the NOTCH-1 heterodimer. This leads to dissociation of NEC and successive metalloprotease cleavage, ubiquination, endocytosis, and cleavage by gamma-secretase, permitting nuclear translocation of the intracellular portion of NOTCH-1 and activation of target genes.

[0027] b) the amino acid sequence of SEQ ID NO:1 is further mutated such that the portion of said amino acid sequence from amino acid 2311 to amino acid 2556 is replaced with a sequence selected from the group consisting of SEQ ID NO:30-SEQ ID NO:59. When mutations are present in both the HD and PEST regions, this leads to receptors in which signaling in response to gamma-secretase is especially enhanced.

[0047] The invention also encompasses a method for determining regression, progression or onset of a pathological disorder characterized by dysfunctional NOTCH-1 signal transduction. The method involves incubating a sample obtained from a patient having or suspected of having the disorder with a complementary nucleic acid hybridization probe having a sequence of nucleotides that is substantially homologous to that encoding one or more mutant NOTCH-1 receptors as described herein. The probe should generally be designed to interact with nucleic acid encoding the mutant form of the receptor and not the normal form as shown in SEQ ID NO:1. Binding between the probe and any complimentary MRNA that may be present in the sample is used to indicate the regression, progression or onset of the pathological disorder in said patient. Alternatively, assays may be performed using antibody (preferably monoclonal antibody) specific for a mutant form of the receptor. As the level of mutant receptor or nucleic acid encoding mutant receptor increases the likelihood that the disorder is present or progressing also increases. Reduced or absent levels of mutant receptor or nucleic acid is an indication of disorder regression or absence.

Problems solved by technology

As a result, the transcription of NOTCH-1-responsive genes continues for an inappropriately long period of time, again, leading to increased signaling and, ultimately, uncontrolled growth.

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