In Vivo Tumor Growth Control by General Control Nonderepressible 2 (GCN2) Targeting Agents Results from Kinase Activation
The objective of this study was to determine the therapeutic effect and PK/PD relationship of Hibercell compound HC-7366 after 3 days continuous BID dosing (7 administrations) in the MOLM-16 subcutaneous leukemia xenograft model. Single point plasma concentrations were determined 1 h post dose on day 4. HC-7366 at dose levels of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg and 30 mg/kg BID dosing all exhibited significant antitumor activities with TGI% values of 74.8%, 91.5%, 93.6%, 82.0%, and 62.0% (P values < 0.05 vs. vehicle control group), respectively. The maximum antitumor effects were achieved at 3 mg/kg which resulted in Partial Response (PR, =50% tumor regression) in 100% of animals. The plasma concentrations of HC-7366 at 1 h post dose on day 4 provided a single point determination of systemic exposure.  The concentrations increased in approximate proportion to dose level across the range 0.3 – 30 mg/kg. These data indicate that HC-7366 treatment exhibits a 'U-shaped' dose/exposure-response relationship in this model when dosed at 0.3, 1, 3, 10 or 30 mg/kg, orally (PO), twice per day (BID). Regarding the safety profile, most animals treated with HC-7366 at dose level of 1 mg/kg and 3 mg/kg showed minor to moderate BW loss during the study period. IHC on these samples demonstrated maximal induction of the ATF4 targets ASNS, PSAT1 and PHGDH at 1 and 3 mg/kg doses, with 0.3, 10 and 30 have smaller increases over vehicle. RNA-Seq confirmed that the ASNS, PSAT1 and PHGDH effects were regulated at the mRNA level in the U-shaped manner. DDIT3, BBC3, and ATF4 mRNA also demonstrated a U-shaped upregulation, with maximal effect at 3 mg/kg. Notably, PPP1R15A, MCL1, PPP1R15B, EIF2AK4, CASP3, and EIF2S1 mRNA showed much more modest regulation. These data strongly suggest that HC-7366 is having its maximal efficacy in MOLM-16 when it is behaving as a GCN2 activator (i.e., at 1 and 3 mg/kg), where higher concentrations begin to inhibit this effect (at 10 and 30 mg/kg). Overall design: RNA-Seq profiling of subcutaneous MOLM-16 xenograft tumor samples from mice treated for 4 days with vehicle as control or 0.3, 1, 3, 10 or 30 mg/kg HC-7366. 9 replicates per treatment arm. Tumor from untreated mice was also collected and profiled (n=5).