In vitro activity: CBL0137 treatment led to complete absence of living cells at concentrations above 2.5 μM. CBL0137 causes a greater reduction in the number of colonies formed of not only MiaPaCa-2 cells when combines with gemcitabine, but also gemcitabine-resistant PANC-1 cells. Treatment of human pancreatic cancer cells with CBL0137 results in a dose dependent reduction of protein and mRNA levels of RRM1 and RRM2

Kinase Assay: MiaPaca2 and BxPC-3 cells were treated with CBL0137, gemcitabine or a combination of the two for 4 or 24h. Cells were harvested in 1x Cell Culture Lysis Reagent containing protease and phosphatase inhibitors. Lysates 5-20 μg were separated on SDS-PAGE gels and transferred to PVDF membranes. Blots were probed with antibodies specific for SSRP1, SPT16, RRM1, and RRM2. GAPDH was used as a loading control. Proteins were visualized using ECL kit.

Cell Assay: Cells were plated in 96 well plates at 10-20% confluency. After overnight incubation, drugs were added to cells as ten 2-fold serial dilutions. Control for no toxicity was 0.1% DMSO and for complete cell death - 50μM solution of 9-aminoacridine. All treatments were done in triplicate. Cell viability was assessed at 72 hrs after start of treatment using Cell Titer Blue Assay. Mean readings from wells with 9-aminacridine were subtracted from all other wells, after that cell viability was calculated at mean reading of three replicates for all treated conditions relative to mean reading of wells treated with 0.1% DMSO.

Patient derived xenografts (PDX) model. Pancreatic tumor surgical samples were obtained with approval from the Institutional Review Board of RPCI. For in vivo efficacy studies, the patient derived tumors were passaged through severe combined immunodeficient (SCID) mice as already described with some modifications. Frozen tumor pieces (8-10mm3) were thawed on ice and after that transplanted into two flanks of 2 donor SCID mice subcutaneously. 2-3 additional tumor pieces were washed in cold PBS and fixed in 10% buffered formalin for H&E and IHC staining. When any of tumors in donor mice reached 500mm3 the tumor was excised from the anesthetized mouse, washed with PBS and cut into 8-10mm3 pieces and re-transplanted in 10 recipient mice. Again part (1/3-1/2) of tumor sample was used for H&E and IHC staining. For combination studies, 2-5 mm pieces were implanted into each flank of SCID mice. For all PDX studies, treatment was started when at least one tumor per mouse reached ~50 mm3. Mice were distributed between groups using alternating method. Mice were monitored daily, weighed 3-5 times per week and tumor size was measured with digital caliper a minimum of twice per week.