Resistance abrogation experiments for PCB197 and S1–12

PCB197 PPTI screen data and S1–12 PPTI screen data were used to generate PTIM models to identify canine and human cross-species mechanistic targets for undifferentiated pleomorphic sarcoma. Consensus targets were chosen for their appearance in human and canine PTIM models; two drugs (obatoclax, an MCL1 inhibitor and panobinostat, a pan-HDAC inhibitor) that most effectively inhibited PTIM-identified blocks at clinically achievable concentrations were selected for validation.

Potential for resistance abrogation by targeting 2 blocks common to both human and canine PTIM models directed a six-arm proof-of-principle experiment to show that inhibition of multiple blocks inhibited could abrogate tumor cell resistance. PCB197 and S1–12 cell cultures were seeded in quadruplicate on 6-well plates (6 plates per cell model) with 10,000 cells per well. Cells were plated 24 h prior to incubation with any drug. The drug concentrations chosen were 1.5 times the EC₅₀ of the PTIM target of interest. The drug selection was based on desired targets, as well as requiring drug concentration for reaching 1.5 times target K_d must also be less than the maximum clinically achievable concentration.

One plate per cell model was assigned to each of the 6 treatment arms: 1) vehicle control; 2) obatoclax for 6 days; 3) panobinostat for 6 days; 4) obatoclax for 3 days, wash, then panobinostat for 3 days; 5) panobinostat for 3 days, wash, then obatoclax for 3 days; 6) obatoclax + panobinostat simultaneously for 6 days. After 6 days, culture plates were washed with PBS and fresh DMEM with 10% FBS was placed in each well. Wells were monitored until confluency was observed. The primary study endpoint was days to well confluency as determined by a single user. Cells were also counted manually with a hemocytometer and photographed to confirm consistency of the user’s definition of confluency. If after 100 days the cells did not reach confluency, the remaining cells are counted and the study concluded. The experimental design and results are available in Fig. 5.

Undifferentiated pleomorphic sarcoma (UPS) Probabilistic Target Inhibition Map (PTIM)-guided resistance abrogation experiments. Values in the center of PTIM blocks represent expected scaled sensitivity following inhibition of associated block targets. a Histology of PCB197 human UPS sample (20x magnification). b Histology of S1–12 canine UPS sample (20x magnification). c Abbreviated PTIM model for the pediatric preclinical testing initiative (PPTI) screen of PCB197 human UPS sample. d Abbreviated PTIM model built from the PPTI screen of S1–12 canine UPS sample. e Schematic of experimental design for resistance abrogation experiments. f Cellular regrowth of PCB197 human UPS sample over 100 days following treatment by single and multi-agent compounds in sequence and in combination. g Cellular regrowth of S1–12 canine UPS sample over 100 days following treatment by single and multi-agent compounds in sequence and in combination. Data in (f-g) is based on n = 4 replicate experiments