Axl Receptor Tyrosine Kinase Signaling Pathway and the p53 Tumor Suppressor Protein Exist In A Novel Regulatory Loop In B-Cell Chronic Lymphocytic Leukemia Cells

Axl is a constitutively active receptor tyrosine kinase (RTK) in CLL B-cells. We have shown that Axl is physically associated with multiple non-receptor cellular kinases and regulates their activities in CLL B-cells including Lyn kinase (a member of Src-family kinases reported to be overexpressed in CLL), PI3K, Syk and phospholipase γ2 (PLCγ2). These aspects likely relate to drug resistance and survival. Importantly, inhibition of Axl by pharmacological inhibitor (R428) induces massive cell death suggesting its critical role in CLL B-cell survival. We hypothesized that in addition to its role in regulating cellular kinases, Axl might modulate the tumor suppressor p53 to make CLL B-cells more resistant to conventional therapeutic agents. This is based on our unpublished findings that CLL B-cells from patients who have undergone treatment without any reduction in tumor burden expressed increased levels of Axl vs. their pre-therapy levels. In addition, we have detected higher levels of Axl in 17p-deleted CLL patients. Thus to understand the mechanism of Axl RTK-mediated regulation of CLL B-cell growth and survival, we interrogated the functional association between Axl and the HDM2 (human homolog of mouse MDM2)/p53 axis.

We cloned the entire (∼2 kb) 3'-untranslated region (UTR) of the Axl transcript into pMIR-Report vector downstream of the fire-fly luciferase gene. Human embryonic kidney cells (HEK293) were transfected with a full length Axl gene containing the first 500 nucleotides of its 3'-UTR or the pMIR-Report plasmid with or without miR-34a. Cell lysates were examined for the expression of Axl by Western blot or performed luciferase assay. Purified CLL B-cells were also treated with various kinase inhibitors including, Wortmannin (pan-PI3K-inhibitor), PP1 (Src-inhibitor) and SKI-606 (targets both Src and Axl) to detect signaling pathway components downstream of Axl involved in regulating HDM2 function.

CLL B-cells from majority of CLL patients (n=15) showed constitutive levels of Axl phosphorylation which we found was associated with the phosphorylation level of HDM2 at Ser-166. HDM2 has an important function in controlling p53 stability in response to genotoxic stress. Recent studies have shown that HDM2 can be phosphorylated on Ser-166 by AKT, MAPKs, and other kinases and this phosphorylation regulates HDM2 nuclearcytoplasmic shuttling under certain conditions. However, we found that HDM2 phosphorylation in CLL is independent of PI3K/AKT, p70S6K, and Erk1/2 activation. Interestingly, we found that inhibition of Axl and/or Src kinases (using SKI-606 and PP1) inhibited HDM2 phosphorylation suggesting that Axl and Src kinases are critically involved in regulating HDM2 affinity towards p53 in CLL B-cells. We also identified a complementary binding sequence of miR-34a microRNA on the first 100 nucleotides of Axl 3'-UTR, suggesting a possible post-translational regulation of Axl. Interestingly, miR-34 family transcription was reported to be regulated by p53, and miR-34a was found differentially expressed in CLL versus normal CD5+ B-cells and is linked to response to therapy in CLL. Indeed, exogenous expression of the Axl gene containing the first 500 nucleotides of the Axl 3'-UTR in HEK293 cells exhibited a dose-dependent down regulation of the Axl protein upon co-transfection with miR-34a using Western blot analysis. Furthermore, activation of p53 in CLL B-cells upon treatment with the DNA-damage agent doxorubicin showed a 4–5 fold decrease of Axl protein by flow cytometry. Activation of p53 by doxorubicin was verified by detecting increased expression of p53 and its acetylation at lysine-382 by Western blot. In addition, luciferase assay data using the Axl-3'-UTR reporter plasmid DNA and miR-34a revealed a dose-dependent inhibition of the luciferase activity. Further studies are underway to fully dissect these novel regulatory signaling pathways. Conclusions: Together, these findings suggest that the two functionally distinct cell growth regulatory pathways can modulate CLL B-cell survival in response to various stimuli. This study has established a link between Axl RTK cell-survival signaling pathway and the tumor suppressor p53-mediated apoptosis-signaling pathway in CLL. These first of a kind observations also underscore that the relative balance of p53 status and Axl levels in tumor cells could relate to CLL therapeutic outcome.

Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.