Cyclin-Dependent Kinase Inhibitors in Hematological Malignancies—Current Understanding, (Pre-)Clinical Application and Promising Approaches

Genetically altered stem or progenitor cells feature gross chromosomal abnormalities, inducing modified ability of self-renewal and abnormal hematopoiesis. Cyclin-dependent kinases (CDK) regulate cell cycle progression, transcription, DNA repair and are aberrantly expressed in hematopoietic malignancies. Incorporation of CDK inhibitors (CDKIs) into the existing therapeutic regimens therefore constitutes a promising strategy. However, the complex molecular heterogeneity and different clinical presentation is challenging for selecting the right target and defining the ideal combination to mediate long-term disease control. Preclinical and early clinical data suggest that specific CDKIs have activity in selected patients, dependent on the existing rearrangements and mutations, potentially acting as biomarkers. Indeed, CDK6, expressed in hematopoietic cells, is a direct target of MLL fusion proteins often observed in acute leukemia and thus contributes to leukemogenesis. The high frequency of aberrancies in the retinoblastoma pathway additionally warrants application of CDKIs in hematopoietic neoplasms. In this review, we describe the preclinical and clinical advances recently made in the use of CDKIs. These include the FDA-approved CDK4/6 inhibitors, traditional and novel pan-CDKIs, as well as dual kinase inhibitors. We additionally provide an overview on molecular mechanisms of response vs. resistance and discuss open questions.

Dual Kinase Targeting in Leukemia

Pharmacological cancer therapy is often based on the concurrent inhibition of different survival pathways to improve treatment outcomes and to reduce the risk of relapses. While this strategy is traditionally pursued only through the co-administration of several drugs, the recent development of multi-targeting drugs (i.e., compounds intrinsically able to simultaneously target several macromolecules involved in cancer onset) has had a dramatic impact on cancer treatment. This review focuses on the most recent developments in dual-kinase inhibitors used in acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), and lymphoid tumors, giving details on preclinical studies as well as ongoing clinical trials. A brief overview of dual-targeting inhibitors (kinase/histone deacetylase (HDAC) and kinase/tubulin polymerization inhibitors) applied to leukemia is also given. Finally, the very recently developed Proteolysis Targeting Chimeras (PROTAC)-based kinase inhibitors are presented.

Design, synthesis, and evaluation of 4,5,6,7-tetrahydrobenzo[d]thiazole-based novel dual kinase inhibitors of CK2 and GSK3β

A rationally designed novel compound (1g) has shown significant dual kinase inhibitory activity against CK2 (1.9 μM) and GSK3β (0.67 μM).

Application of Dual PI-3K/mTOR Inhibitors Is Not Always Superior to Inhibition of mTOR Alone.,

Abstract 3575 ALL cells are highly dependent on bone marrow stromal support for in vitro proliferation and survival. The major regulators of patient-derived ALL cell growth and survival convey their proliferative and survival signals through the phosphoinositide 3-kinase (PI-3K) pathway. It has been recently demonstrated that signalling through PI-3K and AKT is the most important pathway for the proliferative responses of ALL cells to CXCL12, the chemokine predominantly responsible for stromal dependent growth of ALL cells. In addition, inhibition of the mTOR signalling molecule downstream of PI-3K with RAD001 has been shown to inhibit proliferation and induce cell death resulting in extended survival in a NOD/SCID xenograft model of human ALL. This work has supported the initiation of clinical trials of RAD001 in adults with relapsed ALL. Although PI-3K and mTOR have similar and overlapping functions, mTOR can be activated independently of PI-3K, and proliferation and survival can be stimulated by PI-3K in an mTOR independent manner. Therefore combining PI-3K and mTOR inhibition is likely to be advantageous over inhibition of either kinase alone, suggesting disruption of PI-3K/AKT/mTOR signalling will provide a new approach for the treatment of ALL. We investigated the dual kinase inhibitors BEZ235 and BGT226. We have previously demonstrated that PI-3K and mTOR inhibition with the dual kinase inhibitor BEZ235 and BGT226 significantly inhibits the proliferation of ALL cell lines and patient derived stromal dependent lines in vitro, a 3 log greater potency in comparison to the mTOR inhibitor RAD001. The ability to induce cell death differed between the dual mTOR and PI-3K inhibitors. BGT226 potently induced cell death at 1.6μM, while more than 30μM of BEZ235 was required to kill ALL cells within 24 hours, compared to RAD001 which has an IC50 of 16μM. Interestingly, the IC50 for BEZ235 and BGT226 both declined when cell death was measured at 48 and 72 hours, a feature not observed with RAD001. BEZ235 induced extensive caspase 3 cleavage while BGT226 had only a minor effect on the activation of caspase 3. Preliminary data using 3H-thymidine assays, suggests that the addition of dual PI-3K/mTOR inhibitors may not be beneficial with antagonistic interactions being observed with some agents including etoposide and doxorubicin. Further studies will be required to define interactions between kinase inhibitors and chemotherapeutic agents. We also demonstrate the activity of these dual kinase inhibitors in a NOD/SCID xenograft model of human ALL, with treatment commencing once 1% ALL was detected in the blood. The dual kinase inhibitors significantly extended survival in all 4 xenografts compared to control treated animals. However they only produced superior results to RAD001 in one of these xenografts (2032) and were clearly inferior in another (1345). In xenograft 2023 exposure to RAD001 resulted in increased AKT phosphorylation on Ser473, suggesting RAD001 induced activation of mTORC2 through the mTORC1 feedback loop, potentially diminishing responses to mTOR inhibitors. This particularly highlights the benefit of the dual PI-3K/mTOR inhibitors' ability to block mTORC2 signalling through PI-3K inhibition and could possibly reflect the results seen with xenograft 2032 in vivo. The reason for reduced efficacy in xenograft 1345 is also not apparent at this stage but may possibly reflect greater bioavailability of RAD001 as compared to the dual kinase inhibitors. Interestingly, Akt signalling differed between xenografts, suggesting that dependence on PI-3K/Akt signalling upstream and potentially independent of mTOR may impact on the efficacy of the dual PI-3K/mTOR inhibitors however, this does not appear to provide a complete explanation of the different responses observed. Dual kinase inhibitors may offer improved therapeutic outcomes for a subset of ALL patients. More importantly some patients actually respond better to single mTOR inhibitors than dual inhibitors. We need a greater understanding of how these inhibitors work, so that patients that will benefit from dual kinase inhibitors and those where dual inhibitors will be less effective than single mTOR inhibitors can be identified prior to treatment. Disclosures: No relevant conflicts of interest to declare.

Comparision of Dual PI-3K/mTOR Inhibitors with mTOR Inhibitors Using a Pre-Clinical Model of Acute Lymphoblastic Leukemia

Abstract 3258 ALL cells are highly dependent on bone marrow stromal support for in vitro proliferation and survival. The major regulators of patient-derived ALL cell growth and survival convey their proliferative and survival signals through the phosphoinositide 3-kinase (PI-3K) pathway. It has been recently demonstrated that signalling through PI-3K and AKT is the most important pathway for the proliferative responses of ALL cells to CXCL12, the chemokine predominantly responsible for stromal dependent growth of ALL cells. In addition, inhibition of the mTOR signalling molecule downstream of PI3K with RAD001 has been shown to inhibit proliferation and induce cell death. Although PI-3K and mTOR have similar and overlapping functions, mTOR can be activated independently of PI-3K, and proliferation and survival can be stimulated by PI-3K in an mTOR independent manner. Therefore combining PI-3K and mTOR inhibition is likely to be advantageous over inhibition of either kinase alone, suggesting disruption of PI-3K/AKT/mTOR signalling will provide a new approach for the treatment of ALL. We investigated the dual kinase inhibitors BEZ235 and BGT226. Here, we demonstrate that PI-3K and mTOR inhibition with the dual kinase inhibitor BEZ235 significantly inhibits ALL proliferation in vitro, with IC50 values in the range of 7–20nM, indicating a 3 log greater potency in comparison to the mTOR inhibitor RAD001. The ability to induce cell death differed between the dual mTOR and PI-3K inhibitors, with BGT226 potently inducing cell death at 1.6μM, but more than 16μM of BEZ235 was required to kill ALL cells, with a combination of autophagy and apoptosis being detected. While cell death was induced with higher concentrations of BEZ235 than needed to inhibit proliferation, clonogenic assays revealed a major decrease in the survival capacity of cells exposed to the agent. We also demonstrate the activity of these dual kinase inhibitors in a NOD/SCID xenograft model of human ALL with significantly prolonged survival of mice. The potential synergy of dual kinase inhibitors with conventional chemotherapy drugs and in mTOR inhibitor resistant cases remains to be studied. Dual kinase inhibitors may offer an improved therapeutic index through reduced toxicity over mTOR inhibitors, and potentially reduce the risk of development of resistance to kinase inhibition. Disclosures: No relevant conflicts of interest to declare.