Abstract
Axl, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different cancer cells. The Axl ligand growth-arrest specific gene 6 (Gas6) was discovered to promote proliferation of leukemia cells in acute and chronic myeloid leukemia and Axl was identified as a potential therapeutic target in these diseases. Based on these data we investigated the role of Axl in BCR-ABL negative myeloproliferative neoplasms (MPN) and the therapeutic potential of Axl blockade in this group of diseases.
We studied the effects of Axl blockade using the small molecule Axl inhibitor BGB324 and performing a lentivirus shRNA mediated knockdown of Axl in human SET-2 and murine BaF3-Jak2V617F MPN cell lines. Pharmacologic Axl blockade resulted in a significant dose dependent decrease in viability of MPN cell lines as measured by WST-1 cell viability assay. Annexin+ staining revealed an increased rate of apoptotic cells upon BGB324 treatment for SET-2 (increase by 15% at 1µM, p<0.001) and BaF3-Jak2V617F cells (increase by 54% at 2µM, p<0.05). Moreover, Western Blot analysis showed higher levels of cleaved caspase 3 in BGB324 treated SET-2 cells and decreased levels of anti-apoptotic bcl-2 in BGB324 treated BaF3-Jak2V617F cells. Additionally, BrdU incorporation assays showed a dose dependent decrease in proliferating cells upon treatment with BGB324 in MPN cell lines (p<0.05). Genetic knockdown of Axl in SET-2 cells decreased cell viability by 75% (p<0.01), increased apoptosis levels as measured by Annexin+ staining by 61% (p<0.05) and decreased proliferation as measured by BrdU incorporation by 35% (p<0.001) compared to control-transduced cells. Furthermore, Western Blot analysis revealed that genetic knockdown of Axl resulted in decreased phosphorylation of Stat3 and Stat5 compared to control-transduced cells. Combined Axl and Jak2 blockade, using BGB324 and the Jak2-inhibitor ruxolitinib, showed additive effects on reducing cell viability in SET-2 and BaF3-Jak2V617F cells (p<0.01 and p<0.001, respectively). Western Blot analysis identified inhibition of Stat5 by BGB324 single treatment in SET-2 cells whereas additive effects of combined Axl and Jak2 blockade resulted from additional inhibition of Stat3. In BaF3-Jak2V617F cells, BGB324 single treatment resulted in downstream inhibition of Akt signaling whereas additive effects of combined Axl and Jak2 blockade were exerted via additional inhibition of Stat5, Stat3 and Erk.
The finding that BGB324 inhibits growth of MPN cells was further corroborated in vivo. A xenograft tumor model with SET-2 cells was set up in vivo. SET-2 tumor bearing mice treated with BGB324 50mg/kg showed a slower tumor growth (n=8, p<0.01), with a 60% reduction of tumor weight compared to vehicle treated mice (n=8/8, p<0.01). As a second in vivo model, a systemic model of Jak2V617F driven disease was used. After intravenous injection of BaF3-Jak2V617F cells, mice were treated with 50mg/kg BGB324 or vehicle starting the day after inoculation. BGB324 treated mice had a longer overall survival compared to vehicle treated mice (n=10/11, p*<0.05).
Furthermore, to evaluate the potential of BGB324 in primary MPN cells, peripheral blood mononuclear cells (PBMC) were isolated from MPN patients and healthy donors. Western Blot analysis showed higher levels of Axl expression by PBMC from MPN patients compared to PBMC from healthy donors. Moreover, colony-forming assays with PBMC were performed in the presence of different concentrations of BGB324. Here, a higher reduction in the number of colony forming units (BFU-E and CFU-GEMM) was observed in samples from MPN patients compared to healthy donors upon treatment with 1µM (77% vs. 5%, respectively; p<0.001) or 2µM (100% vs. 60%, respectively; p<0.01) of BGB324 (n=5/5).
In conclusion, these data indicate therapeutic potential of Axl blockade in BCR-ABL negative MPN as monotherapy and in combination with Jak2-inhibition, supporting the need for clinical investigation.
Disclosures
von Amsberg: Novartis: Honoraria, Speakers Bureau; Ipson: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau. Loges:BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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