Translational In Vivo and In Vitro Studies in Patients (pts) with Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML) and Myeloproliferative Disease (MPD) Treated with MK-0457 (MK), a Novel Aurora Kinase, Flt3, JAK2, and Bcr-Abl Inhibitor.

Introduction: MK-0457 (VX-680) is a small molecule kinase inhibitor of the Aurora kinases A/B, wild type and mutant (T315I) Bcr-Abl, Flt3, and Jak2 (V617F) with preclinical activity in solid and hematological tumor models. We report correlative laboratory studies from pts with AML, CML and MPD enrolled in a Phase I/II study with MK-0457, on a 5-day continuous intravenous infusion (CIV), dose escalation schedule given at 2 to 3 week intervals.

Methods: Specimens from 6 MPD (all Jak2 (V617F) positive), 6 CML pts in accelerated/blastic phase (3/6 positive for T315I) and 3 AML pts with at least two study time points were assessed for apoptosis (AnnexinV (AV)/PI by FACS) and cell cycle (PI) changes in vivo. A CD34+ progenitor sub-population was studied. PBMC isolated prior to therapy were cultured in vitro with MK-0457, either alone or in co-culture with human, BM-derived mesenchymal stem cells (MSC). Concentration and time dependent apoptosis and cell cycle changes were determined.

Results: At day 5, there was no significant induction of apoptosis in 5/6 MPD pts compared to baseline (BL) (mean for total cell population 37 vs 27%; gated myeloid subpopulation 37 vs 38%). All pts had a hematological response and, except one, a slight decrease in Jak2 (V617F) transcript levels. In vivo, CML pts showed a heterogeneous response. 3/5 pts had an increase (7, 98, 103 %) in AV/PI positive cells and 2/5 pts a decrease (−28, −74%) at day 5, one of which in lymphoid BC, exhibited delayed massive apoptosis at day 11 (470% increase from BL). The other pt without evidence of apoptosis induction achieved a hematological response with disappearance of the T315I clone, relapsing with high Bcr-Abl transcript levels thereafter. A pt with partial cytogenetic and molecular response had high BL apoptosis (50%) that remained high (58%) at 90 days when taken off study due to side effects (muscle cramps/pain). In vitro, a dose dependent rise in specific apoptosis was seen in MPD cells between 48–144 hrs, reaching statistical significance at 50–400 nM MK (p=0.017−0.008, two tailed t-test, unequal variance) and in CML cells at 24, 48, 72 hrs (mean increase at 400nM MK of 17, 14, 28%, respectively in CML, p≤0.05 for mean of all time points). In AML, 2/3 pts had increased numbers of AV/PI positive cells on day 5, declining to BL levels on day 11 in one pt. In vitro, AML specimens (from non-study pts) showed less pronounced apoptosis than MPD and CML cells, with similar effects in CD34 cells. Co-incubation with MSC protected cells of AML and MPD pts at all drug concentrations, reaching significance at ≥100nM in MPD pts (p=0.03). In cell line experiments, HL-60 and OCI-AML cells showed striking G2/M block, polyploidization and apoptosis.

Conclusion: In preliminary experiments, MK-0457 induces apoptosis in AML and CML pts in vivo, shows a time and dose dependent increase of apoptosis in vitro in MPD, CML and AML pts cells and leads to accumulation in G2/M phase. Co-culture with human MSC protect cells from MPD and AML pts in vitro. The clinical trial is ongoing and molecular profiling studies are underway.