DT2216, a Synthetic Proteolytic Selectively Targeting Bcl-X_L for Ubiquitination and Degradation in Tumor Cells but Not in Platelets, Is a Safer and More Potent Antitumor Agent Than Navitoclax

The evasion of apoptosis, or programmed cell death, is a hallmark of cancer, which promotes tumor initiation and progression. The evasion is in part attributable to the over-expression of anti-apoptotic proteins in the Bcl-2 family. In addition, chemotherapy and radiation can upregulate the expression of the Bcl-2 family in cancer cells, which renders them more resistance to cancer therapy. The most common Bcl-2 family member over-expressed in many solid tumor cells and a fraction of leukemia and lymphoma cells is Bcl-X_L and its expression is also highly correlated with resistance to cancer therapy independent of p53 status in many cancers. Therefore, Bcl-X_L is one of the most important validated cancer cell targets.

Inhibition of Bcl-X_L with a small molecule inhibitor has been extensively exploited as a molecularly targeted therapeutic strategy against cancer, resulting in the discovery of several Bcl-2/X_L and Bcl-X_L inhibitors as promising anti-cancer drug candidates including navitoclax. Unfortunately, these inhibitors failed to become anticancer drugs because platelets are also dependent on Bcl-X_L for survival. Therefore, inhibition of Bcl-X_L with Bcl-2/X_L and Bcl-X_L inhibitors causes severe reduction in platelets or thrombocytopenia, an on-target and dose-limiting toxicity, which prevents their use as an effective anticancer drug in clinic. To overcome this problem, we generated a series of novel bifunctional molecules that targeting Bcl-X_L to the ubiquitin-proteasome system (UPS) for degradation. These synthetic proteolytic compounds, termed synthetic proteolytics (Syntholytics) or proteolysis targeting chimeras (PROTACs), were rationally designed to recruit the Von Hippel Lindau (VHL) E3 ligase to ubiquitinate Bcl-X_L for degradation by the proteasome. Because VHL is minimally expressed in platelets, our Bcl-X_L Syntholytics can selectively induce Bcl-X_L degradation in various cancer cells but not in platelets. Amongst these Bcl-X_L Syntholytics, DT2216 was found to be the most potent in inducing Bcl-X_L degradation leading to the loss of viability of Bcl-X_L-dependent T-ALL MOLT-4 cells at nanomolar concentrations but did not cause any platelet toxicity. Compared to navitoclax, DT2216 is more potent in induction of apoptosis in a variety of cancer and leukemia cells in vitro in a caspase-dependent manner. Furthermore, our in vivo studies in immunocompromised mice revealed that DT2216 at 15 mg/kg/wk potently inhibited tumor growth in Bcl-X_L-dependent MOLT-4 T-ALL xenografts as a single agent whereas navitoclax had no significant effect at the same dosage. Dosing with DT2216 at 15 mg/kg every four days significantly regressed larger established MOLT-4 T-ALL tumors that failed to respond to navitoclax treatment.

To assess the therapeutic potential of DT2216 in combination with other Bcl-2 family inhibitors, we employed the Bcl-2/xl dependent NCI-H146 small cell lung cancer cells and the Mcl1/Bcl-xl dependent multiple myeloma EJM cells. The combination of DT2216 with Bcl-2 inhibitor (ABT199) or Mcl-1 inhibitor (S63845) synergistically reduced the viability of H146 and EJM cells, respectively. DT2216 in combination with ABT199 effectively inhibited tumor growth in H146 xenografts. Collectively, our findings suggest that targeting Bcl-X_L using Bcl-X_L Syntholytics can selectively kill Bcl-X_L-dependent T-ALL cells and various solid tumor cells without causing significant platelet toxicity. Moreover, the combination of Bcl-X_L Syntholytics with other Bcl-2 protein inhibitors could be used to effectively target multiple cancer types including both hematological and solid tumors. Therefore, Bcl-X_L Syntholytics have the potential to be developed as safer and more potent novel anti-cancer drugs.

Keywords: Bcl-X_L, VHL, Protein degradation, T-ALL, Cancer, Apoptosis

Disclosures: S.K., X.Z., D.L., Y.H., P.Z., X. L., G. Z., and D.Z. are inventors of a pending patent application for use of Bcl-xl syntholytics as anti-cancer agents. R.H, G.Z. and D.Z. are co-founders of Dialectic Therapeutics that develops Bcl-xl syntholytics.