Glycogen Synthase Kinase-3 (GSK3) Inhibition with Lithium in Combination with Tretinoin Induces Differentiation in Non-Promyelocytic Acute Myeloid Leukemia (AML)

Lithium (Li) is a GSK3 inhibitor that induces differentiation and anti-proliferation of AML cells in vitro.  Pre-clinical studies demonstrated GSK3 inhibition leads to increased expression of the receptor of tretinoin, the retinoic acid receptor.  We conducted a phase I trial of Li in combination with tretinoin for non-promyelocytic AML based on pre-clinical studies demonstrating synergistic AML cell differentiation using the two-drug combination.  We report laboratory correlative data demonstrating AML cell differentiation using the combination of Li and tretinoin. 

Methods.  12 relapsed or refractory AML patients were enrolled.  Li was administered at 300mg three times daily (twice daily in patients age ≥60) continuously and adjusted to meet the target Li plasma concentration of 0.6-1mEq/L; tretinoin doses were 22.5 mg/m²/day (dose level 1) and 45 mg/m²/day (dose level 2) administered on days 1-7 and 15-21 of a 28-day cycle.  Li was given for a 3-day lead-in period prior to cycle 1.  7 patients were evaluable for laboratory correlative studies.  Blood and/or bone marrow samples were collected at baseline, day 1 and day 8 of cycles 1 and 2 and at the end of the study period.  Specimens were processed only if ≥5% leukemic cells were available for analysis.  Immunophenotyping was performed by multi-parameter flow cytometry using fluorochrome-labeled antibodies to CD11b, CD14 and CD15 as markers for monocytic and/or granulocytic differentiation.  Cells were concurrently stained for CD34, CD117 and CD38 for identification of the leukemic blast and stem cell population (CD34+ CD38-).  After fixation and permeabilization using a formaldehyde-based solution, intracellular staining was performed for phosphorylated Serine-9 GSK3β (pGSK3β) as a marker for GSK3 inhibition.  Li directly binds GSK3 and causes reversible enzyme inhibition by Ser9 phosphorylation. 

Results.  The target serum concentration of Li was achieved in all patients and found to inhibit GSK3 in leukemic cells.  pGSK3β measured by mean fluorescence intensity (MFI) increased by a median of 1067 (168-1738) by day 30 in 4 patients with samples available for analysis; the earliest increase in pGSK3β MFI occurred at day 1 after the Li lead-in period.  Six subjects (86%) demonstrated differentiation as defined by ≥10% increase in a cell population expressing at least one of the surface markers associated with monocytic or granulocytic differentiation, with median increase 27.4% (range 10.0-59.2%).  Maximum increase occurred at median day 30 (range 8-60) of the study period.  Three patients (50%) exhibited differentiation in both the CD34+ or CD117+ blast population and in non-blast cells.  Two subjects (33%) had increased differentiation in the non-blast population only, and 1 (17%) had increased differentiation in the blast population only.  Three patients had clinically stable disease (defined as no increase in blasts for ≥4 weeks from study start) and showed a median increase in differentiated cells of 27.4% (17.2-37.6%) compared to a 14.8% (10.0-19.6%) increase in the 3 patients with progressive disease (p=0.44).  Four patients had day 60 bone marrow available for analysis; in 3 patients, there was a median of 29.3% (range 12-32%) increase in cells expressing at least one differentiation surface marker in the CD34+ or CD117+ blast population.  In 6 subjects with evaluable sample, a median relative reduction of 67% (37-98%) of the AML stem cell population (CD34+ CD38-) was seen in the blood at day 8 to 60.

Conclusions.  The combination of lithium and tretinoin induces differentiation in leukemic cells.  Unlike most cytotoxic AML therapies which target rapidly dividing cells, this strategy may be effective in targeting the AML stem cell population.  Response to differentiation therapy was variable, suggestive of the heterogeneity of the disease.  Li induced a modest effect on GSK3 inhibition at clinically achievable plasma concentrations.  Though this study demonstrated the ability to inhibit GSK3 in leukemic blasts and induce differentiation, higher levels of GSK3 inhibition are likely necessary for greater clinical efficacy.  As Li concentrations cannot be increased significantly due to drug toxicity, future studies investigating the use of more potent GSK3 inhibitors in AML are needed to further investigate this strategy.