Previously, the Abl allosteric inhibitor, GNF-2, was shown to cooperate with Imatinib and Nilotinib in inhibiting Bcr-Abl . Furthermore, GNF-2 was also reported to cooperate with oligomerization inhibitors in inhibiting Bcr-Abl, as well as in overcoming T315I resistance . Our results corporate the reported data showing a cooperation between Abl allosteric inhibitor, GNF-2, and Abl ATP competitors (Imatinib and Nilotinib), in inhibiting the proliferation of Ba/F3 cells carrying the native or the T315I mutated Bcr-Abl. However, no cooperation was observed between GNF-2 and AKIs in controlling the proliferation and clonigenicity of the parental Ba/F3 cells (Additional file 1). In this report we also demonstrated that Dasatinib, an Abl/Src dual inhibitor, is capable of cooperating with GNF-2 in inhibiting the proliferation of Ba/F3 cells carrying the native or the T315I mutated Bcr-Abl. In fact, our data showed that the cooperation with Dasatinib was more potent than the one observed with Imatinib or Nilotinib. Furthermore, cooperation between GNF-2 and AKIs was also evident in inhibiting clonigenicity of Ba/F3 cells carrying the T315I mutation of Bcr-Abl. Presence of 1 μM of the AKIs reduced the IC50 of GNF-2 from 25 μM to 10.5 μM, 13 μM, and 3.5 μM when Imatinib, Nilotinib and Dasatinib were used, respectively. Although we did not closely investigate the nature of the cooperation between AKIs and GNF-2 in the current study, it seems that GNF-2 and Dasatinib cooperated in a synergistic manner which is consistent with Mian et al., 2012 finding who demonstrated a synergistic relation between GNF-2 and Dasatinib .
Weisberg et al., 2010 showed that HG-7-85-01, ATP competitor, is capable of inhibiting the Bcr-Abl-T315I gatekeeper mutant. Furthermore, HG-7-85-01 was found to have additive effect in in-vitro and in vivo models in a Bcr-Abl-dependent fashion . Our study utilized GNF-2, and other AKIs that are not active in inhibiting the T315I mutation, resulting in the inhibition of proliferation and clonigenicity of the T315I cells via a mechanism that is not dependent on Bcr-Abl, but rather on an alternative or downstream pathways.
Next, we explored the molecular mechanism responsible for the observed cooperation. Initially, we monitored the cooperation between GNF-2 and suboptimal concentration of AKIs in inhibiting the native Bcr-Abl auto-phosphorylation. Our results, presented in Figure 3, showed a moderate cooperation in inhibiting the phosphorylation of native Bcr-Abl and STAT5α. Our data are in agreement with data generated using flow cytometry analysis illustrating that GNF-5, a GNF-2 analog, cooperated with Nilotinib to inhibit STAT5α phosphorylation . In contrast, only minimal cooperation was seen when Ba/F3 cells carrying the T315I Bcr-Abl was used. These results illustrated that the cooperative inhibition of Ba/F3 cells' proliferation and clonigenicity is not mediated by the Bcr-Abl protein, and that probably the two kinase inhibitors target downstream or alternative signaling pathways that control the growth of these cells.
Results shown in Figures 4 and 5 illustrated that JAK2 is also targeted by GNF-2, however, with reduced potency, consistent with the presence of a myristate binding pocket (MBP) within the JAK2 kinase (data not shown). Moreover, presence of AKIs augmented the inhibitory effect exerted by GNF-2. Interestingly, combination of GNF-2 and Dasatinib was the most efficient combination in inhibiting JAK2 phosphorylation.
Our data are also consistent with findings made by Nelson et al., 2011 showing that inhibitors of alternate pathways, such as STAT5α inhibitors, might be utilized as an effective therapy for Ph+ leukemia carrying native and T315I mutated Bcr-Abl . The enhanced activity of the combination of GNF-2 with Dasatinib, a dual src/Abl kinase inhibitor, might be due to the inhibitory activity of Dasatinib on Src kinase which is involved in STAT5α phosphorylation .
In conclusion, our data provide evidence for cooperation between GNF-2 and AKIs in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl construct. In our experimental system we used a laboratory model of p190 Bcr–Abl, a variant commonly found in acute lymphocytic leukemia (ALL) that typically responds only transiently to AKIs therapy, arguing that ALL patients may benefits from such combination. Cooperation between GNF-2 and AKIs was not mediated by Bcr-Abl protein inhibition, since the phosphorylation levels of Bcr-Abl and STA5α were not affected in Ba/F3 harboring T315I mutated Bcr-Abl. In summary, we showed that drug combination of allosteric inhibitors and AKIs, Dasatinib in particular, allows overcoming resistance in Ph+ leukemia cells, including cells harboring the T315I mutation.