Inhibition of Phosphorylation of ERK in CLL Cells Pre-Treatment Correlates Best with Response to Dasatinib, Fludarabine, and Rituximab for Patients with Relapsed CLL

Survival, proliferation, and resistance to chemotherapy in CLL cells have been consistently shown to be associated with the activity of the B-cell receptor (BCR) and the associated downstream pathways activated by it. Key molecules in this pathway are Lyn and Syk (Spleen tyrosine kinase), as well as PI3K, Btk (Bruton’s tyrosine kinase), and ERK. Dasatinib, given at standard doses, allows for serum levels well above 11 nM, the IC50 for the direct suppression of Lyn kinase and Btk. We have previously shown that dasatinib used as a single agent in patients with relapsed CLL results in lymph node responses in 60% of patients and partial responses in 20% of patients as defined by NCI-WG criteria. In the current study, patients with relapsed CLL were treated with a regimen combining dasatinib at 140 mg/day, days 1-14, with fludarabine (F) 25 mg/m2/day, days 1-3, and rituximab (R) 375 mg/m2 per cycle, repeated every 28 days, for up to 6 cycles. Patients were followed closely for response with CT scans every 2 months initially. Among the first 10 patients treated, the schedule of treatment was altered to determine signal transduction effects and resultant apoptosis of the CLL cells due to the different components of the regimen. Hence, only dasatinib was given on Day 1, only fludarabine and rituximab on Day 3, and all 3 drugs on Day 4. Blood samples were obtained from the patients before dosing and at 6 hours after treatment to measure effects on the CLL cells, which were isolated by standard Ficoll separation and frozen until the assays could be performed. For these 10 patients the median time to progression (TTP) was 21 months, and the initial clinical response was as follows:

Site CR CRi PR SD PD ORR 95% CI

Blood 4 2 4 0 0 100% 74%, 100%

Nodes 6 1 1 1 1 80% 49%, 96%

CT 2 0 2 6 0 40% 15%, 70%

IWCLL 2 0 2 5 1 40% 15%, 70%

Key: CR=complete response in blood = < 4,000/ul lymphocytes,

CR in nodes = no nodes palpable by PE,

CRi = complete response with incomplete blood recovery,

PR=partial response, SD=stable disease, PD=progressive disease, ORR=overall response rate, CI=confidence interval.

IWCLL = International Workshop on CLL criteria.

The patterns of signal transduction in response to the various drugs at 6 hours are shown in aggregate for the patients below. The numbers represent the ratio of phosphorylated protein to total protein at the times indicated with respect to their baseline levels set as 100%. The phosphorylation sites tested were p-Lyn (Y416), p-Syk (Y352), p-ERK1/2 (T202/Y204).

Phosphorylation at 6 h after indicated treatment – % of baseline ± SE (N=7):

Day 1 (D) Day 3 (F+R, no D) Day 4 (D+F+R)

p-Lyn/Lyn: 42% ± 3% 207% ± 63% 58% ± 13%

p-Syk/Syk: 34% ± 15% 122% ± 67% 36% ± 15%

p-ERK/ERK : 64% ± 22% 168% ± 40% 56% ± 22%

The patterns of signal transduction for the 3 patients with the most favorable outcome (TTP and OS) were compared to that for the 4 patients with the poorest clinical outcome. The baseline ratios of phospho-ERK1/2 to ERK1/2 (pre-treatment) correlated most strongly with outcome. Those with a good outcome exhibited low basal p-ERK/ERK (mean 1.0, range 0.1 to 1.8 percent), while patients with a poor outcome exhibited high basal p-ERK/ERK (mean 22.1, range 2.2 to 65.6 percent).

Conclusions: For most patients, dasatinib inhibits (directly or indirectly) phosphorylation of Lyn kinase, Syk kinase, and ERK1/2 in the first 6 hours. The degree of apoptosis (to be presented, but not shown here) resulting from this is variable and is probably affected by activation of the PI3K/Akt pathway and other pathways. The long-term clinical outcome of our patients correlated strongly with the baseline phosphorylation of ERK1/2, suggesting that future treatments of patients with CLL might benefit from targeting ERK directly, or by targeting other molecules in the MAPK pathway.