Cyclin D1 Driven CDK4 Activity Is Responsible for Hypersensitivity to NOXA Inducing Combination Treatment in Mantle Cell Lymphoma (MCL) Cells

Background:

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma with generally poor outcome. Hallmark of MCL is the t(11;14)(q13;q32) chromosomal translocation resulting in overexpression of Cyclin D1 and consequently aberrant activity of CDK4. New molecular targeted drugs such as Palbociclib, an inhibitor of CDK4/6 or Bortezomib, a proteasome inhibitor, have shown promising results in preclinical studies and MCL patients. Our previous research revealed stabilization of the short-lived pro-apoptotic BH3-only protein NOXA as a critical determinant for sensitivity to Bortezomib and other inhibitors of the ubiquitin proteasome system (UPS). We here investigated the role of Cyclin D1-driven abnormal CDK4 activity for NOXA stabilization and induction of cell death upon treatment with UPS inhibitors and identified promising combinations utilizing Cyclin D1-driven CDK4 activity for effective therapy.

Methods:

Experiments were conducted in 4 MCL cell lines (JEKO-1, MINO, Rec1, and JVM-2) as well as in primary cells obtained from peripheral blood samples of MCL patients diagnosed according to the WHO criteria. Bortezomib resistant JEKO-BR cells were obtained by continuous treatment with increasing concentrations of Bortezomib. JEKO-BR gained a cross-resistance to Carfilzomib. Cell death was assessed by flow cytometry staining with AnnexinV-FITC and propidium iodide. Synergy was calculated using CompuSyn Software based on the Chou & Talalay's method. RNA and protein levels were quantified by q-PCR and immunoblotting, respectively.

Results:

Both pharmacological inhibition of CDK4 by Palbociclib and genetic knockdown of CDK4 or Cyclin D1 partially prevented proteasome inhibitor-mediated cell death as well as NOXA accumulation comparable to that observed upon siRNA-mediated NOXA depletion. Inhibition of CDK4 had only minor impact on NOXA (PMAIP1) mRNA expression but significantly reduced proteasome inhibitor-mediated stabilization of NOXA protein, mainly driven by an autophagy mediated proteolysis. In contrast, compounds interfering with NOXA ubiquitination, such as the fatty acid synthase (FASN) inhibitors Orlistat and Cerulenin or the neddylation inhibitor MLN4924, further enhanced NOXA stability upon treatment with the proteasome inhibitors Bortezomib or Carfilzomib leading to super-induction of NOXA protein. This was reflected by a highly synergistic ability to induce apoptosis in MCL cell lines and patient samples (n=11) but had only minor effect on lymphocytes or fibroblasts from normal donors (n=3). Again, induction of cell death as well as NOXA stabilization upon combination of Orlistat together with Bortezomib was antagonized by Palbociclib or by siRNA-driven knockdown of Cyclin D1, CDK4 or NOXA. Finally, to determine if FASN and/or neddylation inhibitors together with proteasome inhibitors may overcome Bortezomib resistance we tested different combinations and found that combining of the 3 compounds Bortezomib, Orlistat and MLN4924 was able to potently kill Bortezomib/Carfilzomib resistant MCL cells. Importantly, this combinatory treatment was still selective and had only minor effects on viability of normal lymphocytes.

Conclusion:

Our data demonstrate that the Cyclin D1-driven CDK4 activity in MCL is important for NOXA stabilization and induction of cell death upon treatment with UPS inhibitors. Importantly, this elevated CDK4 activity also opens the way for effective combination treatment of MCL using ubiquitin modulators together with proteasome inhibitors via super-induction of NOXA protein. On the other hand,particular caution is recommended when combining CDK4 inhibitors together with proteasome inhibitors.