In Vitro and In Vivo Anti-Leukemic Effects of KPT-9274, a Reported PAK4 Allosteric Modulator, in Acute Myeloid Leukemia: Promising Results Justifying Further Development in This Disease

Acute Myeloid Leukemia (AML) is the most common adult acute leukemia and is characterized by numerous driver mutations and/or cytogenetic rearrangements that promote disruption of stem cell/early myeloid progenitor differentiation, apoptosis, and proliferation. Identification of both personalized targets specific to a mutation or genomic abnormality and also global ubiquitous tumor-related targets relevant to AML represents a high priority to improve therapy. p21 protein (Cdc42/Rac)-activated kinase 4 (PAK4) is involved in disease progression for several solid tumors but its expression and contribution to disease pathogenesis in AML has not been examined. Since multiple cellular pathways important in AML (e.g., RAS and Wnt/β-catenin) are regulated by PAKs, we hypothesized PAK4 (and other family members) could represent an attractive pharmacologic target. We first evaluated the expression of PAK4 in AML cell lines and patient blasts (55 patients) using RNA sequencing and Western blot confirmation. This demonstrated PAK4 to be abundantly expressed at the mRNA and protein level in virtually all the analyzed samples. We then tested the in vitro effects of the PAK4 allosteric modulators (PAMs) KPT-9274 (clinical candidate) and KPT-9331 (tool compound) on AML cell lines. These included MV4-11, HL-60, THP-1 and Kasumi-1. Cell lines were treated for 24, 48 and 72 with PAMs KPT-9274 and KPT-9331 at dosages ranging from 1nM to 10uM. Proliferation was measured by MTS assay. All cell lines showed a dose-and time- dependent decrease in cell proliferation with IC₅₀ ranging from 0.14 to 0.28 µM for both compounds. Cell lines with low protein and mRNA expression of PAK4, such as HL-60, were sensitive to PAM treatment suggesting possible alternative targets of these agents. To determine the effect of PAMs on apoptosis, MV4-11 and THP-1 cell lines were treated with KPT-9331 at IC₅₀ concentration (~0.25 µM) and cell death was measured via Annexin-V/PI flow cytometric analysis after 24, 48h and 72h treatment. KPT-9331 induced a time dependent increase in apoptosis in both cell lines. In MV4-11 cells, KPT-9331 caused cell cycle arrest and inhibition of proliferation after 24hrs. We also tested the effect of PAMs in primary AML cells. Patient samples cocultured with a human stromal cell lines were treated with PAMs for 96 hours. IC₅₀ values ranged from 0.14 - 0.19 µM. A dose dependent decrease in proliferation following PAM treatment was observed in all the five analyzed samples irrespective of genetic subtype.

PAMs treatment for 48hrs using a whole blood viability assay from normal donors showed no significant cytotoxic effect on T and NK cells, but modest toxicity to normal B cells. Normal hematopoietic colony forming cell assays are being initiated and will be presented. We next utilized a human AML leukemia xenograft model with MV4-11 cells to assess the in vivo activity of KPT-9274. Mice were dosed once daily via oral gavage with KPT-9274 (150 mg/kg) or vehicle control. KPT-9274 dramatically inhibited tumor growth, prevented invasion of MV4-11 cells, and improved overall survival with all mice (n=7) being alive (median not reached) at day 49 of experiment as compared to 1 out of 7 vehicle-treated mice (median survival 36 days) being alive at this time. In summary, KPT-9274 demonstrates promising activity in pre-clinical AML models and warrants further investigation in this disease. Ongoing efforts include validating the specificity of the reported target in AML (versus alternative targets), in vivo exploration in primary human AML xenograft models, and understanding the effects of this compound on normal hematopoiesis and function.