Joseph Schlessinger, Ph.D

Featured Investigator

Joseph Schlessinger, Ph.D.

Joseph Schlessinger, Ph.D is a world-renowned expert in the molecular anatomy of the FGF receptor family and downstream signaling events mediated by receptor activation. His laboratory has been at the forefront of understanding receptor tyrosine kinase signaling in general and FGF signaling in particular. Using an integrated experimental approach, employing both chemical quench methodology and ESI-LC-MS/MS, and time-resolved ESI-MS, his laboratory has demonstrated that phosphorylation of the catalytic tyrosine kinase domain of FGFR1 occurs through a series of sequential and precisely ordered reactions.  This work has provided the first detailed time sequence for the molecular activation of this receptor. 

With the recognition that FGF signaling plays a critical role in phosphate homeostasis, the Schlessinger lab has turned its efforts in identifying the receptor for FGF-23, the downstream signals from that receptor and how they can be modulated. Together with Dr. Eswarakumar, he has identified a critical role for the FGF receptor substrate, FRS2a in mediating the pathology associated with activating mutations in the 2c isoform of the FGF receptor that lead to premature cranial suture fusion in Crouzon’s syndrome. By mutating the intracellular domain of the Crouzan receptor (Fgfr2c342Y/+) so that it no longer binds FRS2a (Fgfr2c CLR/+ in Fig. 1), he was able to rescue the suture fusion abnormality in these mice (Fig. 1). Of particular interest is his treatment of calvarial explants derived from mice bearing this activating mutation, with a novel tyrosine kinase inhibitor, PLX052 (Fig. 2).

Fig. 1 - Uncoupling of Frs2α from Crouzon-like Fgfr2c mutant (Fgfr2cC342Y /+ ) receptor prevents premature fusion of cranial sutures in mice

Photographs of the heads of 6-week-old live mice (A–C) and Alizarin-stained skulls (D–F). Note the open sutures in the WT and Fgfr2cCLR /+ (rescued) skulls (arrows); microCT scans showing 3D images of calvaria (G–I). Note completely fused coronal suture in Fgfr2cC342Y /+ Crouzon mutant mice (arrows) and open sutures in WT and Fgfr2cCLR /+ mice. (J–L) 2D images of crosssection of coronal suture showing open suture of WT mice, fused suture of Fgfr2cC342Y /+ mice (arrows) and open suture of Fgfr2cCLR /+ mice. (M–O) Histological section of coronal sutures of 1-week-old mice stained with von Kossa and methyl green. Note the presence of bone trabeculae in the sutural mesenchyme in the Fgfr2cC342Y /+ mutant. (Left) WT, (Center) Fgfr2cC342Y /+, and (Right) Fgfr2cCLR /+. C, coronal suture; S, sagittal suture; L, lambdoid suture; F, frontal suture; fb, frontal bone; pb, parietal bone; of, ossification front; me, mesenchyme; and Tb, bone trabecula. [Scale bars: 2 mm (D–I); 100 μm (M–O).]

Fig. 2 - The mechanism of action of FGFR inhibitor revealed by analysis of cocrystal structure in complex with FGFR1 kinase domain

(A) Treatment of 3T3-cells with PLX052 inhibits the kinase activity of FGFR2 and tyrosine phosphorylation of FRS2a in stimulated cells. (B-F) Views of PLX052 bound to FGFR1K. Carbon atoms of FGFR1K and PLX052 are gray and green, respectively. Oxygen atoms are red, nitrogen atoms are blue, and fluorine atoms are magenta. (C-F) The nucleotide-binding loop is shown in blue, the hinge region in magenta, and the catalytic core in yellow. (B) Electron density map of PLX052 bound to FGFR1 kinase domain. (C) Molecular surface representation of PLX052 bound in the cleft between the two lobes of the FGFR1K. (D) Hydrophobic interactions, depicted by dashed lines, between amino acids of the nucleotide-binding domain of FGFR1 and PLX052. (E) Stereo view of the binding of PLX052 to FGFR1K. Solid lines depict hydrogen bonds between PLX052 and amino acids in FGFR1K. (F) Superimposition of PLX052 and AMP-PCP in complex with FGFR1K. View in Right is approximately perpendicular to the left view. Carbon atoms of AMPPCP are colored in cyan and phosphorous atoms in turquoise.

Treatment with PLX052 prevented premature suture fusion but did not interfere with signaling pathways emanating from the wild-type Fgfr2c allele that are required for normal suture development (Fig. 3). 

FGFR inhibitor treatment

Fig. 3 - Suture fusion in calvaria tissue explants is prevented by treatment with FGFR inhibitor

Calvaria harvested from E18.5-day-old embryos of WT (a–h) and Fgfr2cC342Y /+ Crouzon mutant (i–p) mice were cultured either with vehicle (0.2% DMSO) alone (a–d and i–l) or with 1 μM PLX052 (e–h and mp) for 2 weeks. Histological sections of cultured calvaria were made perpendicular to the coronal suture, which passes through the frontal bone (fb) and the parietal bone (pb). Sections were stained with toluidine blue (a, c, e, g, i, k, m, and o), and adjacent sections were stained by von Kossa followed by methyl green counter staining (b, d, f, h, j, l, n, and p). Lower [c, d, g, h, k, l, o, and p (Scale bars: 100 μm)] depicts the higher magnification of the coronal sutures shown in the boxed regions of Upper [a, b, e, f, i, j, m, and n (Scale bars: 500 μm)], respectively. Open (arrow) and fused coronal sutures (arrowhead) are indicated. Fused sutures are observed in untreated calvaria from mutant mice (i–l), and open sutures are observed in calvaria from mutant mice treated with PLX052 (mp).

These findings are particularly germane to the broader issue of attenuating pathologic FGF receptor signaling in hypophosphatemic disorders. In collaboration with Dr. Eswarakumar, he has also found that the Fgfr3c isoform plays a central role in controlling the balance between proliferation and differentiation of chondrocytes during skeletal development and that deficiency of this isoform causes skeletal overgrowth (Fig. 4).

Fig. 4 - Morphometric analysis of the Fgfr3b −/− and Fgfr3c −/− mouse skeletal phenotype

(A–D) Shown are the Alizarin red-stained humerus (A), radius and ulna (B), femur (C), and tibia (D) of 5-month-old WT, Fgfr3b −/−, and Fgfr3c −/− mice. Note the elongation of the bones in the Fgfr3c −/− mice. (E) Histogram of the lengths of the appendicular bones. Values are expressed in millimeters. Sample sizes: WT, n = 10; Fgfr3b −/−, n = 5; Fgfr3c −/−, n = 5. Note the significant difference between WT and Fgfr3c −/− bones.  *p < 0.00001 by paired t test.

Importantly, gain-of-function mutations in Ffgr3 cause the most common form dwarfism in humans, achondroplasia. Dr. Schlessinger’s ability to translate fundamental biological observations into practical clinical therapies is highlighted by his successful launch of three biotech companies, Sugen, Plexxikon and Kholton. These ventures all take advantage of his skill in developing small molecule inhibitors and antibodies for receptor tyrosine kinases. Some of these compounds are now being used to treat advanced hypernephroma as well as a particular form of gastric carcinoma, known as gastrointestinal stromal tumor (GIST).