Chronic lymphocytic leukemia cells from ibrutinib treated patients are sensitive to Axl receptor tyrosine kinase inhibitor therapy

Earlier we have shown the expression of a constitutively active receptor tyrosine kinase Axl in CLL B-cells from previously untreated CLL patients, and that Axl inhibitor TP-0903 induces robust leukemic B-cell death. To explore whether Axl is an effective target in relapsed/refractory CLL patients, we analyzed CLL B-cells obtained from CLL patients on ibrutinib therapy. Ibrutinib-exposed CLL B-cells were treated with increasing doses (0.01- 0.50μM) of a new formulation of high-affinity Axl inhibitor, TP-0903 (tartrate salt), for 24 hours and LD50 doses were determined. Sensitivity of CLL B-cells was compared with known prognostic factors and effect of TP-0903 was also evaluated on Axl signaling pathway in CLL B-cells from this cohort. We detected sustained overexpression of Axl in CLL B-cells from CLL patients on ibrutinib treatment, suggests targeting Axl could be a promising strategy to overcome drug resistance and killing of CLL B-cells in these patients. We found that CLL B-cells from sixty-nine percent of relapsed CLL patients actively on ibrutinib therapy were found to be highly sensitive to TP-0903 with induction of apoptosis at nanomolar doses (≤0.50 μM). TP-0903 treatment effectively inhibited Axl phosphorylation and reduced expression levels of anti-apoptotic proteins (Mcl-1, XIAP) in ibrutinib exposed CLL B-cells. In total, our in vitro preclinical studies showing that TP-0903 is very effective at inducing apoptosis in CLL B-cells obtained from ibrutinib-exposed patients supports further testing of this drug in relapsed/refractory CLL.


Reagents
A high-affinity orally bioavailable Axl inhibitor TP-0903 (tartrate salt) and TP-0903 (free base) was provided by Tolero Pharmaceuticals Inc., Bcl-2 antibody was purchased from BD Pharmingen and antibodies to Actin and Axl were purchased from Santa Cruz Biotechnologies. Tyro3 and MER antibodies were obtained from Abcam and Lifespan Biosciences, respectively. Phosphotyrosine mouse monoclonal antibody (4G10) was obtained from Millipore. All other antibodies were obtained from Cell Signaling Technology.

Treatment of CLL B-cells with TP-0903 and induction of apoptosis
CLL B-cells (2 x 10 6 cells/ml) from CLL patients (previously untreated or ibrutinib exposed) were treated with increasing doses of TP-0903 (either freebase or tartrate salt) (0.01-0.50μM) for 24 hours in serum-free AIM-V media. PBMCs (2 x 10 6 cells/ml) from healthy normals were also treated with increasing doses of TP-0903 (tartrate salt) (0.01-0.50μM) for 24 hours in 10% RPMI medium. TP-0903 treated PBMCs or CLL B-cells were harvested, and induction of apoptosis determined by flow cytometry (FACScan, Becton Dickinson) after staining the cells with Annexin V/propidium iodide (PI) as described [16].

Immunoprecipitation and western blot analysis
For immunoprecipitation (IP) experiments, 0.2-0.3 mg of CLL B-cell lysates was used to IP using appropriate antibodies as described elsewhere [16]. The precipitated immune complex was electrophoresed on SDSpolyacrylamide gels and transferred onto nitrocellulose membranes, and proteins of interest were detected using specific antibodies. CLL B-cell lysates were analyzed for the expressions of Axl, XIAP, Bcl-2, and Mcl-1by Western blot. CLL B-cell lysates were also analyzed for the phosphorylation status of Axl, Btk, AKT and ERK-1/2 using specific antibodies by Western blot.

Reverse transcription polymerase chain reaction (PCR)
Total cellular RNA was extracted using Purelink Mini Kit (Invitrogen) and 1 μg was reverse transcribed using the SuperScript® III First Strand Synthesis Kit (Invitrogen) and PCR was performed using the SYBR Green PCR core Master Mix (Applied Biosystems, Foster City, CA, USA) on a 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. GAPDH was used for cDNA normalization. Btk, Axl and GAPDH primers were purchased from SABiosciences (Frederick, MD, USA).
The study was done using fasting, male Sprague Dawley rats. Both the free base form of TP-0903 and the tartrate salt were formulated in 20% solutol. The free base of TP-0903 was formulated at 5.0 mg/ml (PO) and dosed by oral gavage (18.2 mg/kg). The tartrate salt of TP-0903 was formulated at 6.5 mg/ml to account for the added weight of the salt and dosed by oral gavage (14.5 mg/kg). Plasma samples were taken at 0. 25, 0.5, 1, 2, 4, 8, 12 and 24 h and analyzed for TP-0903 concentration by LC-MS/ MS with reference to a previously determined standard curve. Pharmacokinetic parameters were calculated using a non-compartmental approach with Phoenix WinNonlin 6.3 (Pharsight, Mountain View, CA, USA).

Statistical analysis
CLL prognostic factors (i.e. ZAP70, FISH, IgVH mutation status, Rai stage, CD38, and CD49d) were evaluated for association with TP-0903 apoptosis induction data of the respective CLL B-cells. Associations between prognostic factors and TP-0903 drug sensitivity were computed using the Kruskal-Wallis test for continuous variables and Chi-square test for categorical variables. SAS 9.4 (SAS Institute, Cary, NC, USA) was used for data analysis. The p-values were generated using a paired t-test.