Bayard Clarkson, MD

Dr. Clarkson's laboratory is continuing to dissect the intracellular signaling pathways that are altered by BCR/ABL fusion genes, which are the primary and probably the sole initial genetic abnormalities responsible for the clinical manifestations of chronic myelogenous leukemia (CML), Ph+ALL (acute lymphoblastic leukemia), and chronic neutrophilic leukemia.

CML is a clonal disease initiated in an early hematopoietic progenitor or stem cell that is primarily characterized by a highly consistent reciprocal translocation, t(9;22), resulting in a chimeric BCR/ABL fusion gene that is probably responsible for the manifestations of the initial phase of the disease (i.e., myeloid expansion and multiple, interrelated phenotypic abnormalities that we have collectively designated "discordant maturation"). BCR/ABL fusion proteins have constitutively elevated protein tyrosine kinase activit; and the degree of elevation is directly correlated with the proteins' transforming abilities both in experimental systems and in the severity of clinical manifestations.

Because of this probable causal relationship, we decided to try to identify and characterize the major BCR/ABL substrates and study their interactions. We observed more than a dozen P-tyr proteins that are constitutively phosphorylated in fresh primary primitive CML progenitors that are not detected in comparable primitive normal progenitors. We also noted that 7 of these same proteins are transiently phosphorylated on tyrosine following stimulation of normal progenitors with kit ligand (KL) but not ( or to a much lesser extent) after stimulation by other hematopoietic cytokines. This finding suggested that BCR/ABL kinase may directly or indirectly phosphorylate some of the same P-tyr proteins that are involved in the normal KL/c-kit signaling pathway; thereby distorting the normal transduction of signals in this and related pathways concerned with regulation of proliferation, differentiation, and maturation.

We have now identified all of the major constitutively phosphorylated P-tyr proteins in primary CML blasts and KL-stimulated normal blasts. Two of the most prominently phosphorylated proteins have been identified as novel docking proteins, p62dok-1 and p56dok-2. Both proteins bind to rasGAP when tyrosine is phosphorylated and display additional characteristics of docking proteins, including an N-terminal pleckstrin homology domain and multiple PXXP motifs.

Recently we have identified 2 other P-tyr proteins as SH2-domain-containing phosphatidylinositol polyphosphate 5-phosphatases, SHIP1 (p140), and SHIP2 (p155). Like SHIP1, SHIP2 selectively hydrolyzes the 5'-phosphate from PtdIns(3,4,5)P3 in vitro but, unlike SHIP1, it does not hydrolyze Ins(1,3,4,5)P4. Both P-tyr SHIP1 and P-tyr SHIP2 bind to the PTB domain of SHC but not to its SH2 domain. SHIP2 selectively binds to the SH3 domain of ABL; whereas SHIP1 selectively binds to the SH3 domain of SRC. Moreover, in contrast to SHIP1, SHIP2 does not bind to either the N-terminal or C-terminal SH3 domains of GRB2.

These findings suggest that SHIP1 and SHIP2 may have a different hierarchy of binding SH3-containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and, moreover, that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. There is now good evidence that both P1(3,4,5)P3 and P1(3,4)P2 are implicated in cytokine-mediated signaling; and the observation that both SHIP1 and SHIP2 are constitutively phosphorylated in primary CML progenitors suggests they may have important roles in p210bcr/abl-mediated myeloid expansion.

Protein-protein and protein-phospholipid interactions involved in recognizing and initiating the transduction of signals induced by specific cytokines are extraordinarily complex; as are the molecular interactions involved in regulating the transmission of these signals and governing appropriate cellular responses. The highly specific inter- and intra-molecular binding between different protein domains and with phospholipids allow a remarkable degree of specificity with regard to recognition of specific signals in specific cell types and in ensuring appropriate, measured cellular responses.

Thus it is quite reasonable to expect that the numerous BCR/ABL substrates that are functionally altered by phosphorylation in early CML progenitors may be largely responsible for disrupting multiple, diverse signaling pathways involved in regulation of proliferation, differentiation, maturation, and programmed cell death -- processes that are normally highly integrated and exquisitely coordinated. The task ahead is to identify and fully characterize all the major BCR/ABL substrates, to define their normal functions and interactions and the specific changes caused by BCR/ABL proteins, and finally to identify specific interactions that may prove to be vulnerable targets for developing highly selective therapies.