Cell‐penetrating SH3 domain blocker peptides inhibit proliferation of primary blast cells from CML patients

Abstract

Bcr-Abl contributes prominently to the development of most chronic myeloid leukemias (CMLs). Prior work has identified the adapter protein CRKL as a major substrate of the Bcr-Abl tyrosine kinase. CRKL can also bind via its first SH3 domain [SH3(1)] to specific sequences in Bcr-Abl. Cell-penetrating peptides were developed that bind with high affinity and selectivity to the SH3(1) domain of CRKL. They disrupt Bcr-Abl-CRKL complexes and strongly reduce the proliferation of primary CML blast cells and cell lines established from Bcr-Abl-positive patients. Activation-specific antibodies against phosphorylated MAP kinase (MAPK) showed that MAPK activity is down-regulated in blast cells treated with the CRKLSH3(1) blocker peptides. We conclude that the Bcr-Abl-CRKL complexes are largely dependent on the CRKLSH3(1) domain, that the central mitogenic cascade is down-regulated as a consequence of the disruption of CRKLSH3(1) interactions, and that CRKL therefore contributes to the proliferation of CML blast cells.

Crk family adaptors–signalling complex formation and biological roles

“…These peptides bind with higher a nities than the corresponding peptides completely made up of naturally occurring amino acids. In vivo studies with these compounds are still missing, but it is already clear from the current literature that Grb2 and Crk/CRKL SH3 domain blocker peptides can be carried into cells with peptidevector systems, thereby inhibiting adaptor protein signalling (Cussac et al, 1999;Kardinal et al, 2000Kardinal et al, , 2001Sakkab et al, 2001).…”

“…One possibility to overcome some of the limitations of forced protein expression is the use of small inhibitory molecules which only block the interaction(s) of a single protein domain. Inhibitory molecules for SH2 and SH3 domain binding pockets are currently being developed in the form of cell-penetrating peptides and small compounds (Cody et al, 2000;Kardinal et al, 2000Kardinal et al, , 2001. Their usefulness will critically depend on binding a nities but also on their ability to block with great selectivity only one or very few of the more than 300 SH2 and SH3 domains which exist in human cells.…”

BCR/ABL P190 transgenic mice develop leukemia in the absence of Crkl

“…Di erent experimental approaches have provided evidence that Crkl plays an important role in transducing oncogenic signals of Bcr/Abl (ten Hoeve et al, 1994b;de Jong et al, 1995;Senechal et al, 1996;Heaney et al, 1997;Tari et al, 1997;Kolibaba et al, 1999;Kardinal et al, 2000;Hemmeryckx et al, 2001). The correlation between the consistent and abnormal tyrosine phosphorylation of Crkl and Bcr/ Abl extends itself to transgenic mouse models for Phpositive ALL (de Jong et al, 1995).…”

Philadelphia-positive leukemia: a personal perspective

“…Based on the consistency with which Crkl is found tyrosine phosphorylated by and complexed to Bcr/Abl and its involvement in integrin/cytokine signaling, it was an interesting candidate for a key role in transduction of the oncogenic signals of Bcr/Abl. Moreover, ablation of Crkl protein levels using antisense technology, or blocking the interaction of Crkl with Bcr/Abl through the use of peptides that block binding of the Crkl SH3 domain, is growth-inhibitory for CML blast cells (Tari et al, 1997;Kardinal et al, 2000).…”

“…Tari et al (1997) used three different cell lines established from Ph-positive patients, and the effect of Crkl antisense may relate to the inherently different adaptive properties of cells grown for numerous generations in culture. Kardinal et al (2000) treated peripheral blood-derived blast cells from 16 different CML patients with blocker peptides for 3 days. Of these, 11 showed inhibition of proliferation, with 36 -73% of the cells remaining alive as judged by trypan blue exclusion.…”