Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

The Hsp90 inhibitor IPI-504 rapidly lowers EML4-ALK levels and induces tumor regression in ALK-driven NSCLC models.

Heat shock protein 90 (Hsp90) is an emerging target for cancer therapy due to its important role in maintaining the activity and stability of key oncogenic signaling proteins. We show here that the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion protein, presumed to be the oncogenic driver in about 5% of patients with non-small cell lung cancer (NSCLC), is associated with Hsp90 in cells and is rapidly degraded upon exposure of cells to IPI-504. We find EML4-ALK to be more sensitive to Hsp90 inhibition than either HER2 or mutant epidermal growth factor receptor (EGFR) with an inhibitory concentration (IC)(50) for protein degradation in the low nanomolar range. This degradation leads to a potent inhibition of downstream signaling pathways and to the induction of growth arrest and apoptosis in cells carrying the EML4-ALK fusion. To generate a causative link between the expression of EML4-ALK and sensitivity to IPI-504, we introduced an EML4-ALK cDNA into HEK293 cells and show that the expression of the fusion protein sensitizes cells to IPI-504 both in vitro and in vivo. In a xenograft model of a human NSCLC cell line containing the ALK rearrangement, we observe tumor regression at clinically relevant doses of IPI-504. Finally, cells that have been selected for resistance to ALK kinase inhibitors retain their sensitivity to IPI-504. We have recently observed partial responses to administration of IPI-504 as a single agent in a phase 2 clinical trial in patients with NSCLC, specifically in patients that carry an ALK rearrangement. This study provides a molecular explanation for these clinical observations.

Resolution of abnormal MR signal intensity in patients with stress fractures of the femoral neck.

OBJECTIVE: The purpose of this study was to describe the natural evolution of abnormal MR signal intensity after the diagnosis of a stress fracture of the femoral neck and to ascertain the time to resolution of that abnormal signal intensity. SUBJECTS AND METHODS: Ten patients who had been previously diagnosed with stress fractures of the femoral neck after positive MR scans of the hip were examined with MR imaging at regular intervals. In each patient T1-weighted and short inversion time inversion recovery (STIR) sequences were obtained until the abnormally bright, diffuse MR signal intensity (representing edema) disappeared from the STIR images. Time to resolution was correlated with each patient's age and presence or absence of a fatigue line on MR imaging. Statistical analysis was done using Fisher's exact test. RESULTS: Edema resolved in seven patients within 3 months of initial diagnosis, in two patients within 6 months, and in the remaining patient within 12 months. We found no statistically significant correlation between time to resolution and patient age or the presence of a fatigue line on MR imaging. Residual sclerosis occurred in five patients, all of whom had a fatigue line. Two of these patients developed bright T1 signal (fatty marrow conversion) around the area of sclerosis. In the remaining three patients, STIR images revealed a brightened fatigue line, which we presumed was caused by granulation tissue. CONCLUSION: In this study, 90% of patients showed resolution of abnormal MR signal intensity on STIR imaging within 6 months of the initial diagnosis of stress fracture of the femoral neck. Such data may prove helpful in examining patients with recurrent symptoms who undergo repeated MR scanning. When an abnormally bright, diffuse MR signal intensity on STIR imaging is seen more than 6 months after an original injury, such abnormal signal intensity is likely to represent new injury.