Pexmetinib: A Novel Dual Inhibitor of Tie2 and p38 MAPK with Efficacy in Preclinical Models of Myelodysplastic Syndromes and Acute Myeloid Leukemia

Abstract

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) suppress normal hematopoietic activity in part by enabling a pathogenic inflammatory milieu in the bone marrow. In this report, we show that elevation of angiopoietin-1 in myelodysplastic CD34(+) stem-like cells is associated with higher risk disease and reduced overall survival in MDS and AML patients. Increased angiopoietin-1 expression was associated with a transcriptomic signature similar to known MDS/AML stem-like cell profiles. In seeking a small-molecule inhibitor of this pathway, we discovered and validated pexmetinib (ARRY-614), an inhibitor of the angiopoietin-1 receptor Tie-2, which was also found to inhibit the proinflammatory kinase p38 MAPK (which is overactivated in MDS). Pexmetinib inhibited leukemic proliferation, prevented activation of downstream effector kinases, and abrogated the effects of TNFα on healthy hematopoietic stem cells. Notably, treatment of primary MDS specimens with this compound stimulated hematopoiesis. Our results provide preclinical proof of concept for pexmetinib as a Tie-2/p38 MAPK dual inhibitor applicable to the treatment of MDS/AML. Cancer Res; 76(16); 4841-9. ©2016 AACR.

Fig 1. ANGPT1 is overexpressed in MDS stem and progenitor cells and is associated with worse prognosis in MDS and AML

ANGPT1 expression in 183 MDS bone marrow derived CD34+ cells and 17 healthy control CD34+ cells is shown. (A)(TTest, P Value<0.05). Tie-2 expression in 183 MDS bone marrow derived CD34+ cells and 17 healthy control CD34s is shown (B)(TTest, P Value<0.05). ANGPT1 expression for different subtypes of MDS are shown. RA (Refractory anemia), RARS (Refractory anemia with ringed sideroblasts) and RAEB (Refractory anemia with excess of blasts).(C) (Ttest). Mean percentage of leukemic blast cells in the bone marrow of patients with low and high ANGPT1 expression is shown (D) (Ttest, P Value <0.05). Kaplan Meir curves for patients with AML (N=200)(F) and MDS (N=183)(E) are shown. Log rank P value was 0.001 for MDS and 0.057 for AML cohorts.

Fig 2. Important functional pathways are dysregulated in MDS cases with high expression of Angiopoietin-1

Gene expression profiles from samples with low and high ANGPT-1 were compared and differentially expressed transcripts were identified (FDR<0.1). Genetic pathways controlling Cell Cycle, Hematopoiesis, Cell signaling were dysregulated in high ANGPT-1 cases (A). Gene signature of high ANGPT-1 is similar to known leukemia and healthy stem cell signatures (B).

Fig 3. Knockdown of Tie-2 inhibits proliferation in leukemic cells and leads to enhanced differentiation in healthy human CD34+ cells

Specific knockdown of Tie2 with two different siRNAs was achieved in leukemic KT-1 cell line as demonstrated by qRTPCR (Ttest, P alue<0.05)(A). Proliferation of leukemic cells KT-1 and CMK was assessed by MTT assay after transfection with TIE2 and control siRNAs. Tie2 knockdown led to decreased numbers of viable cells (Ttest, P Value<0.05)(B). Specific knockdown of TIE2 with siRNAs was achieved in primary healthy CD34+ cells as demonstrated by qRTPCR (Ttest, P alue<0.05)(C). CD34+ cells transfected with Tie2 and control siRNAs were grown in methylcellulose for 14 days and demonstrated increased erythroid colony formation (Ttest, P Value<0.05)(D). Colonies were picked and analyzed for differentiation by multiparameter flow cytometry. Increased erythroid differentiation was seen (higher Glycophorin A positivity) after Tie2 knockdown. Increased maturation of erythroid cells was also demonstrated (increased percentages of late erythroid)(E) after specific knockdown of Tie2.

Fig 4. Kinome dendrogram illustrating the selectivity of ARRY-614

The chemical structure of pexmetinib (ARRY-614) is shown (A). Kinome dendrogram illustrating the selectivity of ARRY-614 (B).

Fig 5. Pexmetinib is a potent in vitro and in vivo inhibitor of p38 and Tie-2

HEK-Tie2 cells express high basal levels of p-p38 and were induced to express constitutively active p-Tie-2 by 24 hr preincubation with doxycyclin. Following 2 hr treatment with varying concentrations of pexmetinib, samples were immunoblotted for p-Tie2 and p-p38 then normalized to GAPDH (A). The IC50s for p-Tie-2 and p-p38 inhibition in this mechanistic assay were 16 and 1 nM, respectively (B). Data were combined with results from other in vitro mechanistic models (Angpt-1 or anisomycin-stimulated HUVEC) as well as functional readouts (LPS-induced TNF-alpha release from human PBMCs) and corrected for human protein binding from HWB functional assays. The protein-corrected in vitro IC50s were 2282 nM and 172 nM for Tie2 and p38, respectively (C). To test the prediction, in vivo IC50s were generated from mechanistic (Tie2 and p38 inhibition in HEK-Tie2 xenografts) or functional (inhibition of LPS-induced TNF-alpha release) murine models following a single oral dose of pexmetinib. The in vivo IC₅₀s were 2066 nM and 203 nM for Tie2 and p38, respectively (C).

Fig 6. ARRY-614 abrogates cytokine mediated myelosuppresive effects in hematopoietic cells; inhibits leukemic cell proliferation and stimulates hematopoietic activity in MDS

Leukemic KG1 cells were treated with TNF-α (10ng/ml) with and without ARRY-614 (10uM) for indicated times and immunblotted for phospho/activated p38 MAPK (A). Downstream mediators of p38 MAPK, MAPKAPK2 and EIF4E were also evaluated by immunoblotting (B,C). Human CD34+ cells were grown in methylcellulose media in the presence and absence of TNF-α (5ng/ml) and ARRY-614 (0.1uM). Erythroid (BFU-E) and Myeloid (CFU-GM) colonies were assessed after 14 days. (N=4, TTest). Proliferation of leukemic cells KT-1 and KG-1 was assessed by MTT assay after treatment with ARRY-614. (Ttest, P Value<0.05)(E,F). Primary MDS mononuclear cells (n=6) were grown in methylcellulose with and without ARRY-614. Erythroid (BFU-E) and Myeloid (CFU-GM) colonies were evaluated after 14 days of culture (G). Representative picture of colonies from one sample are shown (H).