Preclinical activity of P276-00, a novel small-molecule cyclin-dependent kinase inhibitor in the therapy of multiple myeloma

Abstract

Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. Here, we sought to identify the effects of targeting cyclin D in MM. We first confirmed cyclin D mRNA overexpression in 42 of 64 (65%) patient plasma cells. Silencing cyclin D1 resulted in 450% apoptotic cell death suggesting its validity as a potential therapeutic target. We next evaluated P276-00, a clinical-grade small-molecule cyclindependent kinase inhibitor as a way to target the cyclins. P276-00 resulted in dose-dependent cytotoxicity in MM cells. Cell-cycle analysis confirmed either growth arrest or caspasedependent apoptosis; this was preceded by inhibition of Rb-1 phosphorylation with associated downregulation of a range of cyclins suggesting a regulatory role of P276-00 in cell-cycle progression through broad activity. Proliferative stimuli such as interleukin-6, insulin-like growth factor-1 and bone-marrow stromal cell adherence induced cyclins; P276-00 overcame these growth, survival and drug resistance signals. Because the cyclins are substrates of proteasome degradation, combination studies with bortezomib resulted in synergism. Finally, in vivo efficacy of P276-00 was confirmed in an MM xenograft model. These studies form the basis of an ongoing phase I study in the treatment of relapsed/refractory MM.

Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma

“…All MM cell lines were cultured as described previously. 28 Fresh PBMCs were obtained from 4 healthy volunteers. BM aspirates from MM patients were obtained after approval from the Massachusetts General Hospital Institutional Review Board.…”

“…After mononuclear cell separation, MM cells were purified by positive CD138 (Syndecan-1) MicroBead selection, as described previously. 29 BM stromal cells (BMSCs) were generated as described previously 28 and incubated in 96-well culture plates (1 ϫ 10 4 BMSCs/well) for 24 hours. After washing, MM cell lines were added to the wells (2 ϫ 10 4 cells/ well) and incubated with medium or with increasing doses of ACY-1215 for the specified times at 37°C.…”

“…Cells were harvested, washed, and stained with annexin V/propidium iodide (PI) as described previously. 28 Annexin V ϩ /PI Ϫ apoptotic cells were enumerated using the Epics flow cytometer. The percentage of cells undergoing apoptosis was defined as the sum of early apoptosis (annexin V ϩ ) and late apoptosis (annexin V ϩ and PI ϩ ) cells.…”

Cyclin-Dependent Kinases (Cdk) as Targets for Cancer Therapy and Imaging

“…Similar to flavopiridol, P276-00 showed antiproliferative and proapoptotic activity in human breast, colon, lung, prostate carcinoma, and promyelocytic leukemia cell lines in vitro (Joshi et al, 2007b). Decreased Rb phosphorylation, G 1 / G 2 phase arrest, and caspase-dependent apoptosis could be observed in preclinical studies with multiple myeloma cells in vitro and in vivo (Manohar et al, 2011;Raje et al, 2009). The purine derivative R-roscovitine inhibits Cdk1, Cdk2, Cdk5, Cdk7, and Cdk9 with selectivity to Cdk4 and Cdk6 (Meijer et al, 1997).…”

Cyclin dependent kinases in cancer

“…Flavopiridol and R-Roscovitine have shown broader activity against CDK2, 7, 9 and RNA polymerase II CTD, leading to the inhibition of transcription. [55][56][57][58] Although selective CDKIs have shown potent cytotoxic activity in MM cells, the underlying mechanism is still unclear.…”