Abstract
Introduction: Many hematological cancers have been successfully treated through identification of specialized targets in each specific tumor subtype (e.g. BTK inhibition in NHL or proteasome inhibition in multiple myeloma). The p21-Activated Kinase 4 (PAK4) is critical to cellular signaling and may represent a new target for therapy in many hematologic malignancies. PAK4 is a member of the PAK family of proteins that regulate cell survival, cell division and apoptosis. The six members of the PAK family are divided into two groups; Group I (PAK1, 2, 3) and Group II (PAK4, 5, 6), based upon their sequence homology and regulatory mechanisms. PAK4 is a member of the group II family of PAKs and is amplified or mutated in many cancer types. PAK4 is also a key downstream effector of the K-Ras pathway.
Methods: Flow cytometry and CellTiter AQueous One (MTS) assays were used to determine compound effects on cell cycle distribution, proliferation and viability. Immunoblots were used to measure effects of compounds on protein steady state levels and phosphorylation. The T-cell ALL cell line, MOLT-4, and the mantle cell lymphoma cell line, Z-138, were used in xenograft models in mice to test the in vivo efficacy of these compounds.
Results: We have identified selective, orally bioavailable, small molecule PAK4 allosteric modulators {PAMs; e.g. KPT-8752 (mw: 585.6), KPT-9274 (mw: 610.6), and KPT-9331 (mw: 628.6)} which demonstrated selective anti-tumor activity in a variety of hematological cancer cell lines (IC50 values = 0.005 – 1 mM). Treatment of cancer cells with these small molecules resulted in the reduction of PAK4 steady state levels and reduced phosphorylation of key growth signaling proteins such as Akt, β-catenin, cofilin, p21, and cyclin D1. There was a measurable increase in phospho-AMPK indicative of autophagy and stress. These allosteric modulators induced apoptosis through the activation of caspases 3 and 8 and subsequent cleavage of PARP. In MOLT-4 and Z-138 xenograft mouse models, daily treatment with oral PAMs resulted in near elimination of small (100 mm3) and large (800 mm3) tumors in the absence of any clinical signs of toxicity within the animals. Additional cell line and primary tumor models are currently being explored.
Conclusions: PAK4 represents a novel anti-cancer target as a major downstream effector of the Ras oncogene. We have identified selective, orally-bioavailable small molecule PAK4 allosteric modulators which induce potent cytotoxicity in multiple leukemia and lymphoma cell lines with minimal toxicity to normal cell in vitro and clear anti-tumor activity with excellent tolerability in in vivo models of hematological cancers. These compounds inactivate PAK4 by directly inducing PAK4 destabilization. This represents a novel mechanism of the protein kinase inactivation involving degradation of PAK4 rather than direct inhibition of the kinase activity. Based on the in vitro and in vivo activity, these PAK4 allosteric modulators show promising results for the treatment of a wide variety of hematological cancers.
Disclosures
Senapedis: Karyopharm: Employment. George:Karyopharm: Employment. McCauley:Karyopharm Therapeutics: Employment, Equity Ownership. Ellis:Karyopharm: Employment. Crochiere:Karyopharm: Employment. Savona:Karyopharm: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics: Employment. Landesman:Karyopharm Therapeutics: Employment. Baloglu:Karyopharm: Employment.
Milvexian, an orally bioavailable, small‐molecule, reversible, direct inhibitor of factor XIa: In vitro studies and in vivo evaluation in experimental thrombosis in rabbits
