Preclinical and early clinical evaluation of the oral AKT inhibitor, MK-2206, for the treatment of acute myelogenous leukemia

Abstract

Purpose: Recent studies suggested that AKT activation might confer poor prognosis in acute myelogenous leukemia (AML), providing the rationale for therapeutic targeting of this signaling pathway. We, therefore, explored the preclinical and clinical anti-AML activity of an oral AKT inhibitor, MK-2206. Experimental Methods: We first studied the effects of MK-2206 in human AML cell lines and primary AML specimens in vitro. Subsequently, we conducted a phase II trial of MK-2206 (200 mg weekly) in adults requiring second salvage therapy for relapsed/refractory AML, and assessed target inhibition via reverse phase protein array (RPPA).

Results: In preclinical studies, MK-2206 dose-dependently inhibited growth and induced apoptosis in AML cell lines and primary AML blasts. We then treated 19 patients with MK-2206 but, among 18 evaluable participants, observed only 1 (95% confidence interval, 0%-17%) response (complete remission with incomplete platelet count recovery), leading to early study termination. The most common grade 3/4 drug-related toxicity was a pruritic rash in 6 of 18 patients. Nevertheless, despite the use of MK-2206 at maximum tolerated doses, RPPA analyses indicated only modest decreases in Ser473 AKT (median 28%; range, 12%-45%) and limited inhibition of downstream targets.

Conclusions: Although preclinical activity of MK-2206 can be demonstrated, this inhibitor has insufficient clinical antileukemia activity when given alone at tolerated doses, and alternative approaches to block AKT signaling should be explored.

Trial registration: ClinicalTrials.gov NCT01253447.

Figure 1. Effects of MK-2206 on AKT signaling in Human AML Cells

(A) OCI-AML3 and U937 cells were exposed to the indicated concentrations of MK-2206 for 6 hours. Whole cell lysates were subjected to immunoblot analyses with the indicated antibodies. (B) Mononuclear cells from AML patients (pt) with high (>50%) blast count were treated with MK-2206 (5 µM) for 24 hours. Expression of AKT signaling proteins was analyzed by immunoblotting (left) and quantified by densitometry (right). Pt#1, refractory AML, N-Ras mutated, del(12p); pt#2, refractory AML, diploid; pt#3, newly diagnosed AML arising from antecedent MDS, N-Ras mutated, del(5q) and -7.

Figure 1. Effects of MK-2206 on AKT signaling in Human AML Cells

(A) OCI-AML3 and U937 cells were exposed to the indicated concentrations of MK-2206 for 6 hours. Whole cell lysates were subjected to immunoblot analyses with the indicated antibodies. (B) Mononuclear cells from AML patients (pt) with high (>50%) blast count were treated with MK-2206 (5 µM) for 24 hours. Expression of AKT signaling proteins was analyzed by immunoblotting (left) and quantified by densitometry (right). Pt#1, refractory AML, N-Ras mutated, del(12p); pt#2, refractory AML, diploid; pt#3, newly diagnosed AML arising from antecedent MDS, N-Ras mutated, del(5q) and -7.

Figure 2. Effects of MK-2206 on cell growth and survival of primary AML cells

Primary AML cells from two AML patients were treated with MK-2206, and effects on apoptosis induction as examined by annexin V staining. Both patients had diploid karyotype; pt#1 had FLT3-ITD mutation, and pt#2 FLT3 D835 gene mutation. The extent of drug-specific apoptosis was assessed by the formula: % specific apoptosis = (test – control) × 100 / (100 – control).(49)

Figure 3. Modulation of protein expression in AML cells by MK-2206 in peripheral blood (PB, 3A) or bone marrow (BM, 3B)

PB or BM mononuclear cells were collected prior to treatment (“1”) or at selected time-points after initiation of MK-2206 (“2”), which was 24 hours for PB samples and at the completion of the 1^st cycle of therapy for BM samples. Protein lysates were subjected to RPPA analysis. Y-axis, normalized log2-transformed relative values of protein expression for the indicated proteins.

Figure 3. Modulation of protein expression in AML cells by MK-2206 in peripheral blood (PB, 3A) or bone marrow (BM, 3B)

PB or BM mononuclear cells were collected prior to treatment (“1”) or at selected time-points after initiation of MK-2206 (“2”), which was 24 hours for PB samples and at the completion of the 1^st cycle of therapy for BM samples. Protein lysates were subjected to RPPA analysis. Y-axis, normalized log2-transformed relative values of protein expression for the indicated proteins.

Figure 4. Immunoblot analysis of PB samples prior to and after MK-2206 exposure in selected patients treated on the trial

Peripheral blood samples were collected before and 24 hours after the first administration of MK-2206 during the initial treatment cycle. Mononuclear cells were separated by Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO) density gradient centrifugation, cells were lysed in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA), transferred to Hybond-P membranes (GE Healthcare, Little Chalfont, United Kingdom), probed with the appropriate antibodies (AKT, Ser 473–phosphorylated AKT, S6 Ribosomal Protein, Ser240/244-phosphorylated S6Ribosomal Protein, 4E-BP1, Thr37/46-phosphorylated 4E-BP1 from Cell Signaling Technologies [Beverly, MA]) and visualized using LI-COR Odyssey system(LI-COR Biosciences, Lincoln, Nebraska). (A) Examples of Western blot for selected AKT targets. (B) Intensity of the immunoblot signals was quantified and the relative intensity compared to total protein (AKT and S6K, respectively) calculated. *Indicates patients with ≥50% decrease in the density of the protein.

Figure 4. Immunoblot analysis of PB samples prior to and after MK-2206 exposure in selected patients treated on the trial

Peripheral blood samples were collected before and 24 hours after the first administration of MK-2206 during the initial treatment cycle. Mononuclear cells were separated by Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO) density gradient centrifugation, cells were lysed in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA), transferred to Hybond-P membranes (GE Healthcare, Little Chalfont, United Kingdom), probed with the appropriate antibodies (AKT, Ser 473–phosphorylated AKT, S6 Ribosomal Protein, Ser240/244-phosphorylated S6Ribosomal Protein, 4E-BP1, Thr37/46-phosphorylated 4E-BP1 from Cell Signaling Technologies [Beverly, MA]) and visualized using LI-COR Odyssey system(LI-COR Biosciences, Lincoln, Nebraska). (A) Examples of Western blot for selected AKT targets. (B) Intensity of the immunoblot signals was quantified and the relative intensity compared to total protein (AKT and S6K, respectively) calculated. *Indicates patients with ≥50% decrease in the density of the protein.