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CDK4 amplification reduces sensitivity to CDK4/6 inhibition in fusion-positive rhabdomyosarcoma

2015-03-25 00:01:52

Clinical Cancer Research; 25 MAR 2015; DOI:10.1158/1078-0432.CCR-14-2955



Mary E Olanich, Wenyue Sun, Stephen M Hewitt, Zied Abdullaev, Svetlana Pack, Frederic G. Barr



Abstract



Purpose:



Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes a PAX3- or PAX7-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in an aggressive subset of fusion-positive RMS. CDK4/6 inhibitors have antiproliferative activity in CDK4-amplified liposarcoma and neuroblastoma, suggesting CDK4/6 inhibition as a potential therapeutic strategy in fusion-positive RMS.



Experimental Procedures:



We examined the biological consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition by LEE011 in fusion-positive RMS cell lines and xenografts.



Results:



Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and non-amplified fusion-positive RMS cells via G1-phase cell cycle arrest. This arrest was mediated by reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, cell cycle distribution, proliferation, or transformation were not observed in RMS cells overexpressing CDK4. Treatment with LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell cycle arrest. Though all fusion-positive cell lines showed sensitivity to CDK4/6 inhibition, there was diminished sensitivity associated with CDK4 amplification and overexpression. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and non-amplified fusion-positive RMS.



Conclusions:



Our data demonstrate that CDK4 is necessary but overexpression is not sufficient for RB-E2F-mediated G1-phase cell cycle progression, proliferation, and transformation in fusion-positive RMS. Our studies indicate that LEE011 is active in the setting of fusion-positive RMS and suggest that low CDK4-expressing fusion-positive tumors may be particularly susceptible to CDK4/6 inhibition.



Introduction






Rhabdomyosarcoma (RMS) is a family of pediatric soft tissue tumors associated with the skeletal muscle lineage. As the most common soft tissue sarcoma of children and adolescents, RMS comprises two major subtypes: fusion-positive and fusion-negative. Although most fusion-positive RMS tumors are characterized by the PAX3-FOXO1 gene fusion, a smaller subset of cases has a PAX7-FOXO1 fusion. Fusion-negative RMS tumors do not harbor recurrent gene fusions. These molecular differences correspond to clinically distinct phenotypes, as fusion-positive (~80% of alveolar) RMS is more aggressive and has an unfavorable prognosis compared to fusion-negative (>95% of embryonal) RMS. This clinical difference is attributable to the propensity of fusion-positive RMS for early dissemination, poor response to therapy, and frequent relapse. The estimated 5-year overall survival rate for fusion-positive RMS cases is ~25% compared to ~75% for fusion-negative tumors, which underscores the need for more effective therapeutic strategies in fusion-positive RMS.



A particularly aggressive subset of fusion-positive RMS tumors was found to harbor amplification of chromosomal region 12q13-q14. This region, which contains the cyclindependent kinase 4 (CDK4) locus, was amplified in ~25% of PAX3-FOXO1-positive tumors and ~4% of PAX7-FOXO1-positive tumors. CDK4 amplification or overexpression occurs in numerous adult malignancies, including breast carcinoma, lymphoma, melanoma, and sarcoma, most notably in >95% of well-differentiated and dedifferentiated liposarcomas. In addition to RMS, CDK4 is also amplified or overexpressed in other pediatric tumor types, such as neuroblastoma.



As one of three interphase CDKs that promote cell cycle progression from G1 to S phase, CDK4 is a well-established proto-oncogene. Upon mitogenic stimulation, CDK4 and CDK6 form active complexes with D-type cyclins and initiate inactivation of retinoblastoma (RB) and related proteins via direct phosphorylation. Phosphorylation of RB proteins results in their dissociation from transcriptional repressor complexes, thereby activating E2F-dependent expression of genes that promote the G1-S-phase transition of the cell cycle and ultimately drive proliferation. The tumor suppressor p16INK4A negatively regulates this signaling cascade by inhibiting assembly and activation of cyclin D-CDK4/6 complexes.



Recent development of a new generation of highly selective small molecule inhibitors targeting CDK4/6 has renewed attention to CDK4/6 inhibition as a potential therapeutic strategy in various tumor types. Three orally bioavailable, selective CDK4/6 inhibitors, including PD0332991, LY2835219, and LEE011, have entered clinical trials, with PD0332991 being the most advanced in development. LEE011 is currently undergoing evaluation as a single agent or in combination therapy in Phase I/II studies in several tumor settings, such as breast cancer (NCT01872260, NCT02088684, NCT01919229), pediatric malignancies, including malignant rhabdoid tumors and neuroblastoma (NCT01747876), and tumors with CDK4/6 pathway activation (NCT02187783). In accordance with previous studies positively associating CDK4 amplification with vulnerability to CDK4/6 inhibition, one criterion for inclusion in the latter trial is amplification of CDK4. This Phase II study underscores the prevailing dogma of CDK4/6-targeted therapies.



Here, we demonstrate that CDK4 is necessary for proliferation and tumor progression but overexpression is not sufficient to increase RB-E2F signaling, cell cycle progression, proliferation, or transformation in fusion-positive RMS. Additionally, we report the first 6 preclinical evaluation of LEE011 in the setting of fusion-positive RMS and propose a novel model of tumor sensitivity to CDK4/6 inhibition in which CDK4 amplification and resultant overexpression confer reduced rather than enhanced susceptibility to LEE011 and PD0332991. Although responses varied, all fusion-positive RMS cell lines evaluated demonstrated sensitivity to CDK4/6 inhibition. Thus, our data support the clinical development of CDK4/6-targeted therapies in this refractory pediatric tumor and suggest that low CDK4-expressing fusion-positive RMS tumors may be especially vulnerable to CDK4/6 inhibition.



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Key Words



rhabdomyosarcoma | PAX3-FOXO1 | CDK4 | LEE011 | CDK4/6 inhibition