Therapeutic targeting of the cyclin D3:CDK4/6 complex in T cell leukemia.

D-type cyclins form complexes with cyclin-dependent kinases (CDK4/6) and promote cell cycle progression. Although cyclin D functions appear largely tissue specific, we demonstrate that cyclin D3 has unique functions in lymphocyte development and cannot be replaced by cyclin D2, which is also expressed during blood differentiation. We show that only combined deletion of p27(Kip1) and retinoblastoma tumor suppressor (Rb) is sufficient to rescue the development of Ccnd3(-/-) thymocytes. Furthermore, we show that a small molecule targeting the kinase function of cyclin D3:CDK4/6 inhibits both cell cycle entry in human T cell acute lymphoblastic leukemia (T-ALL) and disease progression in animal models of T-ALL. These studies identify unique functions for cyclin D3:CDK4/6 complexes and suggest potential therapeutic protocols for this devastating blood tumor.

Prolonged early G(1) arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle-coupled loss of IRF4.

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.

Noxa mediates p18INK4c cell-cycle control of homeostasis in B cells and plasma cell precursors.

Inhibition of Cdk4/Cdk6 by p18(INK4c) (p18) is pivotal for generation of noncycling immunoglobulin (Ig)-secreting plasma cells (PCs). In the absence of p18, CD138(+) plasmacytoid cells continue to cycle and turnover rapidly, suggesting that p18 controls PC homeostasis. We now show that p18 selectively acts in a rare population of rapidly cycling CD138(hi)/B220(hi) intermediate PCs (iPCs). While retaining certain B-cell signatures, iPCs are poised to differentiate to end-stage PCs although the majority undergo apoptosis. p18 is dispensable for the development of the PC transcriptional circuitry, and Blimp-1 and Bcl-6 are expressed fully and mutually exclusively in individual iPCs. However, a minor proportion of iPCs express both, and they are preferentially protected by p18 or Bcl-xL overexpression, consistent with expansion of the iPC pool by Bcl-xL overexpression, or loss of proapoptotic Bim or Noxa. Expression of Noxa is induced during B-cell activation, peaks in iPCs, and selectively repressed by p18. It is required to promote apoptosis of cycling B cells, especially in the absence of p18. These findings define the first physiologic function for Noxa and suggest that by repressing Noxa, induction of G1 arrest by p18 bypasses a homeostatic cell-cycle checkpoint in iPCs for PC differentiation.