(P 099) CONCURRENT INHIBITION OF CDK2 ADDS TO THE ANTI-TUMOR ACTIVITY OF CDK4/6 INHIBITION IN GIST

Objective: Advanced gastrointestinal stromal tumor (GIST) is characterized by genomic perturbations of key cell cycle regulators. Oncogenic activation of CDK4/6 results in RB1 inactivation and cell cycle progression. CDK4/6 inhibitors have been FDA-approved for the treatment of breast cancer, in combination with aromatase inhibitors or estrogen receptor antagonists. A clinical trial failed to demonstrate any clinical activity of palbociclib monotherapy in advanced GIST, indicating that CDK4/6 inhibition alone is inadequate for disease control in this population. Herein, we use genomic sequencing and multiplexed protein imaging to detect co-dysregulation of the CDK2 and CDK4/6 pathways in GIST clinical specimens. We evaluate strategies of therapeutic cell cycle restoration through combined inhibition of CDK2 and CDK4/6 and define mechanisms of acquired CDK inhibitor resistance in GIST.

Methods: Targeted next-generation sequencing and multiplexed protein imaging were used to detect cell cycle regulator aberrations in GIST clinical samples (N=18), including 8 metastatic TKI-resistant GISTs. Multiple metastases were analyzed in 3 patients. The impact of inhibitors of CDK2 (CDK2 inhibitor-II, PF-07104091), CDK4/6 (palbociclib, abemaciclib), and CDK2/4/6 (PF-06873600) was determined through cell proliferation and protein detection assays in vitro and in vivo. Mechanisms of acquired CDK2 and CDK4/6 inhibitor resistance were characterized in GIST cell lines after long-term exposure and validated through functional studies in vitro and in vivo.

Results: The results demonstrate recurrent genomic aberrations in cell cycle regulators causing co-activation of the CDK2 and CDK4/6 pathways in clinical GIST samples. Identical aberrations of p16, RB1, and p53 were present in all metastases from 3 patients. We show that therapeutic co-targeting of CDK2 and CDK4/6 is synergistic in GIST cell lines with intact RB1, through inhibition of RB1 hyperphosphorylation and cell proliferation (P< 0.01). Intact RB1 predicted response to treatment, whereas RB1-deficient models were resistant. Moreover, we identify RB1 inactivation and a novel oncogenic cyclin D1 resulting from an intragenic rearrangement (CCND1::chr11.g:70025223) as mechanisms of acquired CDK inhibitor resistance in GIST. The CCND1 rearrangement deleted the cyclin D1 C-terminal Thr286 and Thr288 residues which mediate cyclin D1 proteasomal degradation, resulting in overexpression of an abnormal cyclin D1. CDK inhibitor resistance properties were corroborated by lentiviral transduction of the CCND1 fusion gene into fusion-negative GIST, leiomyosarcoma, and breast cancer cells. A modified construct with restored C-terminal properties was used to rescue cyclin D1 protein degradation.

Conclusion: These studies establish the biologic rationale for CDK2 and CDK4/6 co-inhibition as therapeutic strategy in patients with advanced GIST, including metastatic GIST progressing on tyrosine kinase inhibitors. In addition, these findings expand the spectrum of potential CDK inhibitor resistance mechanisms with translational potential for improving cell cycle targeted therapies in other cancer types.