ALBERT

Description: BioTheryx's small-molecule kinase inhibitor, BTX-A51 (the ditosylated salt of A51), has recently received FDA approval of its IND application to initiate a Phase I clinical trial in relapsed or refractory acute myeloid leukemia (AML). A51 (API of BTX-A51) is a multi-kinase inhibitor that blocks the leukemic stem cell target, Casein Kinase 1α (CK1α), as well as the super-enhancer regulator, Cyclin-Dependent Kinase 9 (CDK9), thus preventing the transcription of key oncogenic genes. This molecule has demonstrated remarkable preclinical animal efficacy inferring the eradication of AML stem cells and its potential for use in treating multiple malignancies. To exploit the unique properties of this multi-kinase inhibitor in the context of kinase protein degradation, Proteolysis-Targeting Chimeras (PROTACs) of A51 were investigated. PROTACs utilize the cell's natural ubiquitin-proteasome system to induce the selective and sustained degradation of unwanted disease-causing proteins. PROTACs are heterobifunctional molecules which are comprised of an E3-ubiquitin-ligase ligand which is covalently linked to a target-protein ligand. Through concomitant binding to an E3 ligase and to a target protein, a PROTAC promotes ubiquitination and ultimately degradation of the target protein via the proteasome. As a reversible kinase inhibitor, A51 binds stoichiometrically to CK1α and CDK9 and requires continuous occupancy of these proteins to sustain its unique inhibitory activity. A PROTAC of A51, however, would act catalytically and ablate the target proteins. In this study, PROTACs were assembled through chemically linking A51 to BioTheryx's proprietary Protein Homeostatic Modulators (PHMs™). PHMs™ are a new class of small molecules which bind to the E3 ubiquitin ligase, Cereblon (CRBN), and promote proteasomal degradation of known as well as unreported clinically-relevant CRBN neo-substrates. For the discovery of PHM®-A51 PROTACs, CDK9 crystal structure (6GZD.pdb) and CRBN crystal structure (5FQD.pdb) were used independently for computational drug design. Upon testing in AML and lymphoma cell lines, the PHM®-A51 PROTACs rapidly induced the degradation of both CK1α and CDK9. Additionally, these PROTACs had diminished effect on the protein levels of other A51 targets while inducing the degradation of other desirable targets unaffected by A51. This distinct selectivity arises from the PROTACs' ability to facilitate formation of a ternary complex that enables subsequent ubiquitination of the bound target protein - a selectivity not inherent to any kinase inhibitor. Further, the PHM®-A51 PROTACs have demonstrated low-nanomolar inhibition of cell proliferation in both AML and lymphoma cell lines yet have significantly less toxicity in fibroblast cells and PBMCs. In addition, this class of PROTACs has good pharmaceutical properties, as demonstrated by pharmacokinetic studies in mice, and will advance into AML and lymphoma in vivo studies. The targeted degradation of CK1α and CDK9 using safe, bioavailable drugs designed using strategically-selected PHMs™ with unique biological properties of their own is a very promising approach to treating hematological malignancies and other devasting cancers. Disclosures No relevant conflicts of interest to declare.

Description: Cancer stem cells present a major therapeutic challenge - their effective eradication depends on therapeutic targeting of the relative vulnerabilities of the cancer stem cells vs. normal tissue stem cells. One feature that distinguishes cancer initiating and propagating cells from their normal counterparts is transcriptional addiction, providing opportunities for novel therapeutic interventions with the aim of curing cancer. CKIα ablation appears as a promising means of activating p53 and killing leukemia cells in MDS and AML (Elyada et al , doi:10.1038/nature09673; Kronke et al, doi: 10.1038/nature14610); yet, with no selective CKIα inhibitors for in vivo use, the therapeutic value of CKIα inhibition cannot be validated. We succeeded in developing two different classes of CKIα inhibitors, one inducing CKIα degradation, and another targeting the catalytic pocket of CKIα, with no degradation. Both inhibitor types activate p53, yet we found that CKIα ablation or degradation was not sufficient to selectively eliminate leukemia stem cells, due to robust expression of anti-apoptotic oncogenes driven by super-enhancer activation. Through extensive medicinal chemistry, we obtained CKIα catalytic inhibitors with profound killing capacity of primary mouse AML cells and human AML cell lines. We demonstrated a strong therapeutic effect in a mouse model of MLL-AF9-induced leukemia, with 〉40% cure rates evident by transplantation of bone marrow from treated leukemic mice to lethally irradiated normal mice. All transplanted mice regained normal blood counts, exclusively derived from the donor bone marrow. None showed any evidence of residual disease for 6 months, demonstrating a successful therapeutic window, distinguishing leukemia stem cells (LSCs) from HSPCs. Parallel in vitro experiments, show that the inhibitors did not affect normal hematopoietic progenitors in a CFU assay, but eliminated AML progenitors at an IC50