The Degree of Response of Philadelphia Chromosome Positive Acute Lymphatic Leukemia to Allosteric Inhibition by GNF-2 Depends on the Presence of Either p185-BCR/ABL or p210-BCR/ABL
Authors: Mian Afsar , Metodieva Anna , Najajreh Yousef , Serve Hubert , Ottmann Oliver , Mahajna Jamal , Ruthardt Martin 
Published: October 2019

Abstract
Abstract 598
The t(9;22) translocation (Ph+) leads to the formation of the chimeric bcr/abl fusion gene, which encodes the BCR/ABL fusion protein. Depending on the minor and major breakpoint (m-BCR or M-BCR, respectively) on chromosome 22, either the p185-BCR/ABL (p185) or the p210-BCR/ABL (p210) fusion protein is expressed. In contrast to p210, p185 lacks the putatively oncogenic dbl- and pleckstrin homology domains. In the majority of the cases Ph+ ALL patients harbor the p185, but with increasing age the number Ph+ ALL patients with the p210 increases up to an 40%. Only few functional and biological differences between p185 and p210 are known. Both exhibit a constitutively activated kinase activity responsible for the induction of the leukemic phenotype in contrast to their physiological counterpart, c-ABL, whose kinase activity is finely regulated. One reason is that BCR/ABL “escapes” auto-inhibition mechanisms of c-ABL, such as the allosteric inhibition by “capping”. “Capping” is the process by which myristoylation of the N-terminal cap region of c-ABL leads to an auto-inhibited conformation through its binding to the hydrophobic myristoyl binding pocket (MBP) in the kinase domain. The cap region of c-ABL is encoded by the exon I, which is replaced by the N-terminus of BCR in the fusion protein. Myristate “mimicks” and MBP binders, such as GNF-2, aim to restore the allosteric inhibition through the binding to the MBP in BCR/ABL. The aim of our study was to further develop allosteric inhibition in Ph+ ALL. Therefore we investigated the anti-proliferative activity of GNF-2 in different Ph+ leukemia models such as patient derived Ph+ lymphatic cell lines, long term cultures of Ph+ ALL patient derived bone marrow cells (LTC) and factor dependent lymphatic Ba/F3 cells retrovirally transduced with either p185 or p210. Here we report that i.) the IC50 of GNF-2 for p210-positive ALL cells were much lower (cell lines =125nM; LTC from two different patients 100–300nM) than that for p185-positive ALL cells (cell lines=500 nM; LTC from two different patients 500–1000nM); ii.) factor independent growth of Ba/F3 cells expressing p185 was inhibited with an IC50 of 750nM whereas that of Ba/F3 cells expressing p210 was abolished already at 125nM; iii.) 1000nM GNF-2 was able to abolish colony formation in soft agar of p210-positive but not of p185-positive LTC; iv.) increasing concentrations of GNF-2 abolished in p210-positive ALL cells the BCR/ABL kinase activity, whereas in p185-positive cells it only reduced it as shown by the effects of GNF-2 on the autophosphorylation (Y245) and substrate phosphorylation (Crkl) activity as well as on down stream signaling (Stat5) of BCR/ABL.
Our here presented data provide first evidence of a differential response of p185- and p210-positive Ph+ ALL cells to the allosteric inhibition by GNF-2, as a expression of functional and biological differences between the two fusion proteins within the same disease entity. It remains to clarify not only the mechanisms of this differential response, such as three dimensional conformation, different kinase activity and related signal transdcution, but also its prognostic significance for patients with p210-positive Ph+ ALL.

Disclosures:
No relevant conflicts of interest to declare.