Targeting the p27^Kip1 E3 Ubiquitin Ligase for the Treatment of Multiple Myeloma and Other Hematological Malignancies.

Introduction: The proteasome inhibitor bortezomib (VELCADE®; formerly PS-341) has been approved for therapy of relapsed/refractory multiple myeloma but may be limited in its effectiveness by activation of an anti-apoptotic heat shock protein response. In contrast, targeting a unique E3 ubiquitin ligase could selectively stabilize specific cellular proteins whose ubiquitination is regulated by this E3, reducing the likelihood of any unwanted pro-survival effects on other pathways.

Methods: To this end, we sought to identify a small molecule that preferentially stabilized the tumor suppressor proteins p21^Cip1 and p27^Kip1.

Results: Here we report the identification of CC-125007, a small molecule that stabilized p21^Cip1, p27^Kip1, and p57 in models of multiple myeloma. Unlike bortezomib, CC-125007 did not induce accumulation of other cell cycle regulatory proteins such as p53. CC-125007 reduced viability in a time- and concentration-dependent fashion and, again unlike bortezomib, which induced arrest at G₂/M, CC-125007 induced cell cycle arrest at the G₁/S phase. Decreased viability due to CC-125007 was not accompanied by caspase-mediated apoptosis, however, since it could not be blocked by pre-incubation with a pan-caspase inhibitor. Instead, a puncate monodansylcadaverine staining pattern was observed, demonstrating the formation of autophagic vacuoles consistent with activation of autophagy, or type II programmed cell death. Further supporting this possibility, combination of the autophagy inhibitor bafilomycin A1 and CC-125007 diminished autophagic vacuole formation. Furthermore, Western blotting demonstrated that CC-125007 induced conversion of microtubule-associated protein light chain 3 form I to II, a biochemical marker for autophagy. Meanwhile, Western blotting also showed that CC-125007 did not activate the anti-apoptotic heat shock response proteins 27, 70, and 90, which would be typical of bortezomib. CC-125007 overcame melphalan and bortezomib resistance in multiple myeloma cell lines, and a combination of low dose bortezomib and CC-125007 triggered synergistic anti-myeloma activity. Importantly, in freshly isolated bone marrow mononuclear cells purified from multiple myeloma patients, CC-125007 decreased the viability of the CD138⁺ population to a much greater extant than that of the CD138⁻ cells. CC-125007 also stabilized p27^Kip1 in samples from patients with acute myeloid leukemia, and enhanced bortezomib-induced apoptosis. Studies to elucidate the mechanism by which CC-125007 stabilized p27^Kip1 indicate direct targeting of the E3 ligase.

Conclusions: Together, these data suggest that p27^Kip1 E3 ligase inhibition with agents such as CC-125007 is a rational strategy for the treatment of multiple myeloma and other hematological malignancies, providing a framework for further development of this novel class of agents.