A Clinical Trial of the STAT3 Inhibitor Pyrimethamine in Chronic Lymphocytic Leukemia

Despite advances in targeted therapy, chronic lymphocytic leukemia (CLL) remains a highly prevalent and essentially incurable malignancy. One molecular hallmark of CLL is the constitutive serine phosphorylation and transcriptional activation of the oncogenic transcription factor STAT3. Targeting STAT3 may have a high therapeutic index since normal cells can tolerate a loss of STAT3 function. To identify STAT3 inhibitors that could be rapidly introduced into proof-of-concept clinical trials, we screened a chemical library of drugs known to be safe in humans for specific inhibitors of STAT3-dependent transcription. Using this strategy, we identified the anti-parasitic agent pyrimethamine as a drug that could inhibit STAT3 at levels safely achieved for months at a time in humans. Pyrimethamine inhibited STAT3-dependent gene expression in CLL cells ex vivo, and decreased survival of CLL cells, but not peripheral blood mononuclear cells from healthy donors. To determine whether STAT3 inhibition would confer clinical benefit in patients with CLL, we conducted a phase I clinical trial of continuous daily pyrimethamine, in the era before targeted therapy, in relapsed CLL patients whose disease progressed despite standard therapies. We used a typical 3+3 dose escalation design with three cohorts, 12.5, 25 and 50 mg per day (mg/d). Samples for PK/PD analysis were drawn weekly in the first month and every other week in the second. Sixteen heavily pretreated patients enrolled on the phase 1 portion of this study. The median age was 63 (36-85) and the median time from diagnosis to study therapy was 74 months (range, 6-176 months). Six patients had 17p deletion and 4 had 11q deletion, while 12 of 13 evaluable patients had unmutated IGHV. The patients had a median of 6 prior therapies, including 8 patients who had received prior high dose methylprednisolone, five who had received prior alemtuzumab, and one each with prior allogeneic and autologous stem cell transplantation. Three patients each enrolled on cohorts 1 and 2, and ten patients enrolled on cohort 3. There were no dose limiting toxicities and no significant drug-related toxicities. No objective responses by IW-CLL 2008 criteria were observed. Half of patients achieved stable disease, with one patient dosed at 50 mg/d on therapy for 12 months, and two at 25 mg/d on therapy for 4 and 6 months. The remaining patients had progressive disease, and all but one patient discontinued therapy for progressive disease. The median time on therapy was 1.1 months (0.23-9.99), with median progression free survival of 1.5 months (0.92-5.52) and median overall survival of 22 months (11.75-NA).

To determine whether the levels of pyrimethamine achieved in patients corresponded with levels known to inhibit STAT3 transcriptional function in vitro, we measured trough concentrations of pyrimethamine in plasma and in peripheral blood mononuclear cells at distinct time points for patients on the trial. Plasma concentrations and intracellular concentrations were highly correlated, and also correlated closely with dose level. Only in the highest dose cohort did the plasma concentrations reproducibly approach the threshold of 10 micromolar at which STAT3 inhibition occurred consistently in vitro.

To determine whether pyrimethamine was exerting an on-target effect in inhibiting STAT3-dependent gene expression in vivo, we first defined a signature of five STAT3-dependent genes (AIM2, ATXN1, ENPP2, GAB1, and ID3) that are upregulated in CLL cells relative to normal B lymphocytes. We then determined expression of these genes from the patients' CLL cells at baseline and while on pyrimethamine. In addition, to further generate predictors of response, we also quantitated the cytotoxic effect of pyrimethamine on the pre-treatment cells of patients ex vivo. We then correlated these potential predictive and pharmacodynamic biomarkers with drug levels and clinical course.

In conclusion, pyrimethamine is an inhibitor of STAT3 transcriptional function that can safely be given to patients with CLL. It may be necessary to increase the daily dose of pyrimethamine to beyond 50 mg/day to adequately determine whether this drug can exert an on-target therapeutic effect in CLL patients. Since STAT3 target genes include many immune-suppressive and anti-apoptotic genes, it is likely that pyrimethamine will be most beneficial when combined with other therapeutic modalities.