Targeted Axl Inhibition Primes Chronic Lymphocytic Leukemia B Cells to Apoptosis and Shows Synergistic/Additive Effects in Combination with BTK Inhibitors

Abstract

Purpose: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite aggressive therapeutic approaches. We previously found that Axl receptor tyrosine kinase (RTK) plays a critical role in CLL B-cell survival. Here, we explored the possibility of using a high-affinity Axl inhibitor as a single agent or in combination with Bruton's tyrosine kinase (BTK) inhibitors for future clinical trial to treat patients with CLL.

Experimental design: Expression/activation status of other members of the TAM (e.g., Tyro3, Axl, and MER) family of RTKs in CLL B cells was evaluated. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 with or without the presence of CLL bone marrow stromal cells (BMSCs). Inhibitory effects of TP-0903 on the Axl signaling pathway were also evaluated in CLL B cells. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination.

Results: CLL B cells overexpress Tyro3, but not MER. Of interest, Tyro3 remains as constitutively phosphorylated and forms a complex with Axl in CLL B cells. TP-0903 induces massive apoptosis in CLL B cells with LD50 values of nanomolar ranges. Importantly, CLL BMSCs could not protect the leukemic B cells from TP-0903-induced apoptosis. A marked reduction of the antiapoptotic proteins Mcl-1, Bcl-2, and XIAP and upregulation of the proapoptotic protein BIM in CLL B cells was detected as a result of Axl inhibition. Finally, combination of TP-0903 with BTK inhibitors augments CLL B-cell apoptosis.

Conclusions: Administration of TP-0903 either as a single agent or in combination with BTK inhibitors may be effective in treating patients with CLL.

Figure 1. CLL B-cells overexpress Tyro3 and associates with Axl

A. Tyro3 is overexpressed in CLL B-cells. Lysates from purified CLL B-cells (P1–P8) and normal B-cells obtained from healthy individuals (N1–N5) were examined for the expression of Tyro3 by Western blot analysis using a specific antibody. Actin was used as loading control. CLL patients and normal individuals are indicated by assigning arbitrary numbers. B. Tyro3 is constitutively phosphorylated in CLL B-cells. Total tyrosine-phosphorylated proteins were immunoprecipitated from purified CLL B-cell lysates used above as indicated using a phospho-tyrosine specific antibody (4G10) followed by Western blot analysis using a specific antibody to Tyro3. Presence of immunoglobulin G (IgG) heavy chain was used as loading control C. CLL B-cells co-express constitutively phosphorylated Axl and Tyro3. Tyrosine-phosphorylated proteins were immunoprecipitated from CLL B-cell lysates using 4G10 antibody and phospho-Axl or phospho-Tyro3 was detected in Western blot analysis using a specific antibody to Axl or Tyro3, respectively. CLL patients are indicated by assigning arbitrary numbers. D. Axl and Tyro3 form a complex. Tyro3 was immunoprecipitated from lysates of CLL B-cells using an antibody to Tyro3, followed by Western blot analysis to detect Axl. The blot was stripped and reprobed with an antibody to Tyro3. IgG heavy chain was used as loading control. CLL patients are indicated by assigning arbitrary numbers. Molecular sizes are indicated using standard molecular weight protein markers (Bio-Rad).

Figure 2. Impact of Axl inhibition on CLL B-cell survival

A. Axl expression level is positively associated with its phosphorylation status. Axl was immunoprecipitated from equal amount of lysates of CLL B-cells expressing low levels of Axl (≤20%) or high levels (≥60%) as determined by flow cytometric analysis. The immune complex was then analyzed for the presence of phosphorylated Axl in Western blot using 4G10 antibody. The blot was stripped and reprobed with an anti-Axl antibody. IgG heavy chain was used as loading control. B. Axl inhibition induces robust apoptotic cell death. CLL B-cells conferring low-risk FISH detectable chromosomal abnormalities or not from previously untreated CLL patients (n=20) were treated with increasing doses of an orally bioavailable high affinity Axl inhibitor TP-0903 for 24 hours. Cells were harvested, stained with annexin V-FITC/PI and analyzed on flow cytometer to determine total apoptotic cell death. Results are presented as mean values with standard deviations (SD) at each dose of TP-0903. The mean LD₅₀ (~0.14 μM) value is indicated by the arrow. C. Impact of Axl inhibition on high-risk CLL B-cell survival. CLL B-cells of high-risk as determined by FISH detectable chromosomal abnormalities at chromosome 17p13.1 (n=8) or 11q22.3 (n=10) were treated with increasing doses of TP-0903 for 24 hours. Apoptosis induction was determined as described above. Results are presented as mean values with SD. Arrows indicate mean LD₅₀ values of TP-0903 for the CLL cohorts. D. Relation of Axl expression levels and sensitivity of CLL B-cells to TP-0903. Axl expression on CLL B-cells was determined by flow cytometric analysis using a specific antibody to Axl prior treatment with TP-0903 as described above (panels B&C). LD₅₀ doses for individual CLL samples were calculated from the dose-response curve and plotted against % Axl expression. E. Impact of TP-0903 on normal immune cells. PBMC isolated from normal, healthy individuals (n=5) were treated with increasing doses of TP-0903 (0.01–0.5μM) and cultured in serum-free AIM-V medium for 24 hours. Apoptosis induction was determined as described above. Results are presented as mean values with SD. F. TP-0903 targets phosphorylated Axl in CLL B-cells. Axl was immunoprecipitated from lysates of CLL B-cells treated with a sub-lethal dose of TP-0903 (0.1μM for 20–24 hours), followed by Western blot analysis to detect the levels of phosphorylation on Axl using 4G10 antibody. The blot was stripped and probed with an antibody to Axl. IgG heavy chain was used as loading control. G. TP-0903 does not target P-Tyro3 in CLL B-cells. Tyrosine-phosphorylated proteins were immunoprecipitated from the above TP-0903 treated CLL B-cell lysates, followed by Western blot analysis to detect Tyro3 using a specific antibody. IgG heavy chain was used as loading control. CLL patients are indicated by assigning arbitrary numbers.

Figure 3. Impact of TP-0903 treatment on Axl downstream targets, caspase 3 activation and PARP cleavage

A. TP-0903 reduces phosphorylation on AKT/Src kinase and expression of anti-apoptotic proteins. DMSO or TP-0903 treated CLL B-cell lysates used in Fig. 2F were analyzed for the status of AKT and Src phosphorylation as downstream signaling mediators of Axl by Western blots using specific antibodies to P-AKT or P-Src. Respective blots were stripped and reprobed with AKT or Src antibody. Status of anti-apoptotic proteins Bcl-2, XIAP and Mcl-1 were also analyzed in these TP-0903 treated CLL B-cell lysates in Western blots using specific antibodies. Actin was used as loading control. B. Depletion of Axl in MDA-MB-231 cells reduces expression of XIAP and Bcl-2, but not Mcl-1. MDA-MB-231 cells were transfected with a siRNA targeted to Axl or sc-siRNA as control. After 48 hours of transfection, cell lysates were prepared and expression of Axl, XIAP, Mcl-1 and Bcl-2 was analyzed in Western blots using specific antibodies. Phosphorylation status of AKT (Ser-473) was also examined by Western blot analysis. The blot was stripped and reprobed to detect total AKT. Actin was used as loading control. C. TP-0903 reduces phosphorylation on FOXO3a and upregulates BIM expression. DMSO or TP-0903 treated CLL B-cell lysates used above (panel A) were further analyzed for the phosphorylation status of FOXO3a, a downstream target of AKT and an upstream transcriptional activator of the BIM gene, in Western blots using a specific antibody. Expression of the pro-apoptotic protein BIM was also examined by Western blot using a specific antibody which recognizes both the large (BIM_L) and short (BIM_S) forms of BIM. Actin was used as a loading control. D. TP-0903-induced apoptosis involves caspase 3 activation and PARP-cleavage. DMSO or TP-0903 treated CLL B-cells used above were further analyzed for the activation of caspase 3 and PARP-cleavage by Western blots using specific antibodies. CLL patients are indicated by assigning arbitrary numbers.

Figure 4. TP-0903 overcomes CLL BMSC mediated protection of CLL B-cells from apoptosis

A. Impact of TP-0903 on CLL B-cells in co-culture with CLL BMSCs. Purified CLL B-cells sensitive to TP-0903 were cultured alone or co-cultured with CLL BMSCs from 3 different CLL patients and treated with a dose (0.1μM) of TP-0903 which is lower than the mean LD₅₀ dose (0.14μM) or a dose (0.175μM) of TP-0903 which is higher than the LD₅₀ dose for 24 hours. CLL B-cells cultured alone were also treated with the above doses of TP-0903. Induction of apoptosis in CLL B-cells was determined by flow cytometric analysis using annexin V/PI staining and presented as percent viability. B. TP-0903 does not exert cytotoxic effect on BMSCs. CLL BMSCs co-cultured with CLL B-cells above were harvested following treatment with TP-0903 and analyzed for apoptosis induction as described above. Results are presented as percent viability of the cells.

Figure 5. Combined effect of Axl and BTK inhibition on CLL B-cell survival

A. Combined treatment of CLL B-cells with TP-0903 and BTK inhibitors augments apoptosis levels. CLL B-cells from different CLL patients were treated with increasing doses of TP-0903, ibrutinib or TP-4216, a reversible BTK inhibitor, as a single agent or in combination of TP-0903 with ibrutinib or with TP-4216 as indicated. After 24 hours, cells were harvested and induction of apoptosis was determined as described elsewhere. Results are presented as mean values with standard deviations. B. Combined treatment of CLL B-cells with TP-0903 and ibrutinib shows moderate cytotoxic effects. Combination index (CI) of the results obtained from individual CLL samples following treatment with TP-0903 and ibrutinib as presented in panel A was calculated following the method of Chou and Talalay. CI values <1 indicate a synergistic effect, CI value of 1 indicates additive effects and values >1 indicate antagonistic effects of combined treatment. C. Administration of TP-0903 with a reversible BTK inhibitor TP-4216 has more effective combination effects. CI values of the results obtained from combined administration of TP-0903 and TP-4216 on CLL B-cells from individual CLL patients were calculated similarly as described above. CLL B-cells from majority of CLL patients show additive/synergistic effects (6 of 10) to the combined treatment with TP-0903 and TP-4216. D. BTK inhibitors target phosphorylated BTK in CLL B-cells. Purified CLL B-cells were treated with DMSO, ibrutinib (0.75μM) or TP-4216 (0.75μM) for 24 hours. Cells were harvested, lysed and examined to detect expression of P-BTK (Y223) in Western blot using a specific antibody. The blot was stripped and reprobed with an antibody to BTK. β-tubulin was used as loading control. CLL patients are indicated by assigning arbitrary numbers.