The observation of glioblastoma (GBM) acquiring resistance to treatment is a well-established clinical reality. Increased understanding of GBM adaptive mechanisms to specific therapies is important for achieving improved treatment outcomes for patients, by targeting adaptive responses of tumors. Herein, we investigated GBM xenograft adaptation to sustained cdk4/6 inhibition, which we have previously shown to inhibit the growth of p16 deficient GBM xenografts. To understand GBM adaptation to sustained cdk4/6 inhibition, mice with intracranial U87 xenografts were administered daily PD0332991. Resected tumors from three mice, at time of required euthanasia from tumor burden, were transplanted subcutaneously into the flanks of mice that continued to receive daily PD0332991. At 34 to 145 days of continuously daily treatment, subcutaneous tumors began to grow rapidly, suggesting tumor adaptation to sustained treatment. One of the subcutaneous tumors, beginning to grow rapidly while being subjected to daily PD0332991 treatment, was resected and used in developing a cell suspension that was intracranially injected in a series of mice for analysis of tumor response to PD0332991 by bioluminescence imaging and survival analysis. Cells from resected, PD0332991-resistant tumor were used for in vitro propagation and associated analysis of cell cycle distributions, and for the activation state of cell proliferation. Mice intracranially injected with PD0332991-resistant U87 cells, and receiving continued daily treatment with PD0332991, experienced no survival benefit relative to untreated mice bearing the same tumors, supporting our development of a PD0332991 resistant derivative of U87. Interestingly, tumors established from PD0332991 resistant cells showed more rapid intracranial growth than tumors established from previously untreated U87 cells, resulting in significantly reduced survival of mice bearing PD0332991 resistant tumors. In vitro, PD0332991-resistant cells did not exhibit growth arrest upon exposure to PD0332991, whereas previously untreated U87 cells arrested when treated with PD0332991. Expression analysis of cell cycle regulatory proteins by western blotting revealed that PD0332991 resistant cells were deficient in Rb protein expression, in contrast to control U87, being positive for Rb expression. FISH and PCR revealed deletion of RB gene as being responsible for the lack of Rb protein in PD0332991 resistant U87. Furthermore, PD0332991 resistant cells were examined using RTK arrays, revealing PD0332991 resistance as being accompanied by increased activation of Akt signaling. Our results suggest the potential of p16 deficient tumors to adapt to sustained PD0332991 treatment by genetic inactivation of RB. Moreover, concurrent inhibition of Akt, in treating p16 deficient tumors, could possibly prevent tumor adaptation to cdk4/6 inhibitor therapy.

Citation Format: Yu-Jen Lu, Man-Tzu Wang, Todd Waldman, Tomoko Ozawa, David James. In vivo modeling identifies RB1 suppression and inactivation as being responsible for acquired PD0332991 resistance in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 690. doi:10.1158/1538-7445.AM2014-690