CDK6 degradation hits Ph⁺ ALL hard

In this issue of Blood, De Dominici et al have identified and tested cyclin-dependent kinase 6 (CDK6)-selective proteolysis-targeting chimeras (PROTACs) that suppressed Philadelphia chromosome-positive (Ph⁺) acute lymphoblastic leukemia (ALL) ex vivo and in mice more effectively than approved kinase inhibitors of CDK4/6.¹ 

PROTACs represent a new paradigm in pharmacology with the potential to be as transformative for cancer treatment as targeted kinase inhibitors, therapeutic antibodies, or immunotherapies.²  PROTACs are bifunctional molecules that use 1 arm to bind a protein target and the other to bind an E3 ubiquitin ligase. The ligase then labels the target with a polyubiquitin chain, thereby marking it for degradation by the cell’s disposal machinery: the proteasome. It is the fundamentally different pharmacology of PROTACs that may be the key to its success. For conventional drugs, a high systemic drug exposure is needed to ensure sufficient occupancy of drug binding sites in vivo. In contrast, PROTACs act catalytically and iterative, meaning that after inducing degradation of its target, the PROTAC molecule can be reused for further rounds of target binding and degradation. Therefore, the efficiency of PROTACs may be less susceptible to resistance development that is caused by increased target expression or mutations of the target protein. Impressive progress was made: in only 5 years, PROTACs to >50 distinct target proteins, including many key oncogenes in hematological disorders, were developed and successfully validated in proof-of-concept studies in cell culture and animal models.³  The broad investment of the biotechnology and pharmaceutical industries has resulted in rapid PROTAC drug development and the initiation of the first clinical trial of a PROTAC degrader targeting the androgen receptor in patients with prostate cancer in March 2019.⁴ 

De Dominici et al have developed a PROTAC targeting CDK6 in Ph⁺ ALL. CDK6 was previously validated as a drug target in Ph⁺ ALL that may be exploited in cases of drug resistance.⁵  CDKs are key cell-cycle regulators. CDK4 and CDK6 are ubiquitously expressed, bind d-type cyclins, and drive proliferation by relieving the transcriptional repression of E2F-dependent genes by phosphorylating the retinoblastoma protein. This drives cells through the G1 phase into the S phase of the cell cycle. The partially overlapping and redundant physiologic roles of CDK4 and CDK6 were studied in great detail and unexpectedly identified a kinase activity–independent role of CDK6 as a transcriptional regulator mediating growth-promoting functions.⁶  Thereby, targeted degradation of CDK6 by means of a PROTAC may be able to kill 2 birds with 1 stone through blocking functions of CDK6 that are dependent on its catalytic activity, as well as its independent functions (see figure). Those cannot be targeted with conventional drugs, including the potent Food and Drug Administration–approved small molecule CDK4/6 kinase inhibitors, palbociclib, ribociclib, and abemaciclib, which are approved for the treatment of patients with hormone receptor–positive breast cancer.⁷  Neutropenia, observed in approximately two thirds of these patients and caused by concomitant CDK4 and CDK6 inhibition in hematopoietic progenitors, and CDK4/6 inhibitor resistance due to compensatory increases in CDK6 expression are some of the shortcomings of palbociclib treatment. Hence, selective ablation of CDK6 expression with a PROTAC strategy would target both the kinase activity-dependent and -independent functions of CDK6 and possibly diminish adverse events and resistance development (see figure).

To provide a solid basis for a possible therapeutic application of a CDK6 PROTAC in Ph⁺ ALL, De Dominici et al first compared a genetic CDK6 knockdown with palbociclib treatment side by side. In Ph⁺ ALL cell lines ex vivo and a mouse xenograft model, CDK6 knockdown induced more apoptosis, stronger reduction in leukemic burden, and marked longer survival when compared with palbociclib treatment. Analysis of gene expression profiles identified candidate genes that were differentially regulated by CDK6 silencing and palbociclib treatment. Importantly, several of these genes also correlated with CDK6 expression in samples from Ph⁺ ALL patients. The authors then synthesized and carefully evaluated several derivatives of palbociclib linked to either cereblon or VHL, the most commonly used E3 ligases for PROTAC approaches. YX-2-107 was developed and resulted in rapid degradation of CDK6, but surprisingly not CDK4, and inhibition of CDK6 signaling, in line with previous reports on other CDK6 PROTACs.^8-10  Selective CDK6 degradation was also observed in normal hematopoietic progenitors, but did not change the cell-cycle distribution of these cells. For a possible clinical translation, the authors demonstrated excellent bioavailability and plasma stability of YX-2-107, which contributed to strong inhibition of leukemic burden in mouse xenograft experiments with Ph⁺ ALL cell lines, as well as comparable or superior effects in patient-derived xenografts with sensitive and TKI-resistant patient cells when compared with palbociclib treatment. Importantly, YX-2-107 did not perturb normal mouse hematopoiesis. Apart from Ph⁺ ALL, other diseases with CDK6 dependence, such as other subtypes of B-cell ALL, acute myeloid leukemia, multiple myeloma, and mantle cell lymphoma, may benefit from the superior activity CDK6 PROTACs as compared with palbociclib.

Given that palbociclib is equally potent in inhibiting CDK4 and CDK6 kinase activity, a striking molecular finding was that YX-2-107, which contains palbociclib as a targeting moiety, efficiently degraded CDK6, whereas CDK4 protein levels remained unchanged. This further supports prior evidence that PROTACs may have increased selectivity as compared with their parent drug. Therefore, besides the “added value” of PROTACs to degrade its target on top of its inhibition, it may also improve the therapeutic window by being more selective with less adverse events caused through inhibition of off-targets. If this would turn out to be a general feature of PROTACs, drug candidates who have failed in clinical trials due to safety concerns may be revived and have a second life as a PROTAC.