PMC

(A) Chemical structure of VX-970. (B) Exponentially growing H2009 cells were treated overnight (17 h) with the indicated concentrations of VX-970 alone (lanes 1-6) or in combination with 20 μM cisplatin (lanes 7-12). Cells were then harvested and analyzed for the expression of P-Chk1-S345, P-H2AX-S139 and P-KAP1-S824 by immunoblotting.

Dissociated tumor cells were treated in triplicate in vitro with a matrix of VX-970 and cisplatin concentrations, and synergy or antagonism was analyzed at the 95% confidence interval with MacSynergy II software. Degree of synergy, shown as positive log volume, and antagonism, shown as negative log volume, are shown.

(A-G) Human primary tumor tissues were passaged in SCID mice. Treatment started when the average tumor size was approximately 200 mm³. Tumor bearing mice were treated with vehicle, VX-970 (30 mg/kg in all models except 60 mg/kg in OD26749 and OD26131) PO, 4 consecutive days a week, alone and in combination with cisplatin (3 mg/kg IP, q7d), and cisplatin alone. Tumor volume and body weight were measured twice a week. Studies were terminated one or two days after the final dose of VX-970. Points show the mean tumor volume (mm³) for each treatment group (n=5-10).

(A) Analysis of shifts in the concentration of DNA damaging agent required to inhibit cell viability by 50% (IC₅₀) was used to determine the synergistic or antagonistic effects of a DDR inhibitor. Heat map representing the IC₅₀ shift with the largest absolute value observed with VX-970 or AZD7762 in combination with cisplatin, etoposide, gemcitabine, oxaliplatin and irinotecan across a panel of 36 lung cell lines at 96 h. The colors represent a shift range from −10 (antagonism-blue) to +10 (synergy-red). (B) Histograms showing the percentage of cell lines with > 3-fold (top panel) or 10-fold (bottom panel) synergy with VX-970 (ATR) or AZD7762 (Chk1/2) in combination with cisplatin, oxaliplatin, irinotecan, gemcitabine and etoposide. (C) Impact of p53 on response to VX-970 in A549 cells. Histogram depicts maximum IC₅₀ shift in vector control and p53 knockdown cells observed with VX-970 in combination with cisplatin, etoposide, gemcitabine, oxaliplatin and irinotecan.

(A) Mice bearing OD26749 tumors were treated with a single dose of VX-970 (60 mg/kg PO) and cisplatin (3 mg/kg IP) either as monotherapy or in combination. P-Chk1 and P-H2AX were analyzed by western blot 4 and 48 h after treatment, respectively, and were corrected to total H2AX levels. (B) Representative PET/CT images of mice at baseline (top row) and six days following the indicated treatment (bottom row). Concentration of ¹⁸FLT is indicated by the intensity of the orange color in the PET image. An X-ray CT image of the skeletal system (white) is superimposed for anatomical reference. The tumor is circled (green). Treatment with VX-970 alone had no discernable effect on PET signal (image not shown). (C) Average standard uptake value (SUV) of ¹⁸FLT. (D) Tumor size changes as determined by CT. (E) Examination of prognostic value of ¹⁸FLT imaging. (F) Assessment of the durability of response to VX-970. OD26749 tumors were passaged in SCID mice to P4. Treatment started when the average tumor size was approximately 200 mm³. Tumor bearing mice were treated with vehicle, VX-970 alone (60 mg/kg PO, 4 consecutive days a week), cisplatin alone (3 mg/kg, IP, q7d) and the combination, for two weeks. Vehicle and VX-970 groups were terminated after the final dose of VX-970. Following treatment discontinuation, tumor growth in the cisplatin alone and combination groups was followed twice a week until the average tumor volume reached 1000 mm³. The arrowhead on the graph marks the end of treatment. Error bars are standard errors for all graphs.