Investigation of a Novel Cyclin-Dependent-Kinase (CDK) Inhibitor Cdki-73 As an Effective Treatment Option for MLL-AML

Abstract

The Acute Myeloid Leukemia (AML) subtype characterised by translocations of the Mixed-Lineage Leukemia gene, MLL (t11q23; MLL-AML), is a particularly devastating disease with a median overall survival of only 9 months with current standard therapy. Cyclin dependent kinase (CDK) 9 inhibitors (CDK9i) directly target the CDK9/cyclin T complex (pTEFb) that is essential for activity of the MLL-fusion proteins and for transcriptional elongation, and therefore leads to reduction of transcript levels for multiple key leukemic oncogenes e.g. HOXA9, MYC and MCL1. Several observations suggest that utilising CDK9i to simultaneously target these oncogenes will be an effective strategy for AML, and MLL-AML in particular: (i) Leukemic stem cell (LSC) fractions of AML cells express a high level of MCL1, (ii) Targeting MCL1 has been demonstrated to reduce leukaemia cell survival in a murine model of MLL-ENL, (iii) MCL1 is consistently elevated in AML patients at relapse, (iv) HOXA9 is critical for leukemogenesis in many AMLs, in particular MLL-AML, (v) MYC has been shown to be a critical oncogene in MLL-AML, and (vi) CDK9 function has been shown to be important for MYC-driven tumorigenesis. Our in vitro and in vivo data support the clinical potential of a novel orally bioavailable inhibitor of CDK9, CDKI-73, as an effective therapy for MLL-AML patients. CDKI-73 is a potent inhibitor of CDK9 (Ki 3.5nM)1 and has been shown to induce down-regulation of MCL1, and cell death of Chronic Lymphocytic Leukemia (CLL) B-cells2 and Ovarian Cancer (OvCa) cells3 with nanomolar potency. At doses that are highly toxic for tumour cells, CDKI-73 shows limited toxicity for normal T- and B- Lymphocytes, Bone Marrow Mononuclear cells (BMMNC) and normal colony forming cells (CFC) from the BMMNC fraction. CDKI-73 has many favorable properties also making it an excellent clinical candidate for AML when compared to other CDK9i; in particular, CDKI-73 is (i) unique in its spectrum of inhibition, including targeting CDK6 (IC50 = 0.038 µM; a critical kinase for MLL-AML4), and is (ii) orally bioavailable (F = 56%)2, facilitating sustained in vivo target inhibition. Here we present data showing that in MLL-AML cell lines, CDKI-73 induces growth suppression and apoptosis associated with rapid loss of Myc and MCL1, and activation of PARP. In primary AML patient samples treated with 200nM CDKI-73, we have observed a similar decrease in MCL1 protein levels, with increased 7AAD uptake and Annexin-V staining, consistent with apoptotic cell death. Using a subcutaneous MV4;11 nude mouse xenograft model, we have shown that oral dosing of CDKI-73 (100 mg/kg once every 3 days for 18 days) resulted in a high level of anti-tumour efficacy (p<0.0001 compared to vehicle-treated mice), with minimal toxicity. Moreover, for an established MLL-AML patient-derived xenograft (PDX) generated in NOD/SCID-IL2RG-/- (NSG) mice we also observed significant inhibition of human AML in peripheral blood (p<0.0001), BM (p<0.05) and spleen (p<0.001) with administration of CDKI-73 at 75 mg/kg every 3 days for 15 days. In both models CDKI-73 was well-tolerated at these doses, consistent with our published and preliminary data showing differential effects of CDKI-73 on tumour versus normal cell populations. Given this data, our priority now is to establish the effectiveness of CDKI-73 across a larger panel of primary MLL-AML samples, in further patient derived AML xenografts, and as a combination treatment with AML chemotherapy. REFERENCES: 1. Shao H, Shi S, et al. (2013). J Med Chem. 56(3):640-59. 2. Walsby E, Pratt G, et al. (2014). Oncotarget. 5(2):375-85. 3. Lam F, Abbas AY, et al. (2014). Oncotarget. 5(17):7691-704. 4. Placke T, Faber K, et al. (2014). Blood. 124(1):13-23. Disclosures No relevant conflicts of interest to declare.

The Progression of Acute Myeloid Leukemia from First Diagnosis to Chemoresistant Relapse: A Comparison of Proteomic and Phosphoproteomic Profiles

“…A similar kinase–substrate prediction scenario was observed in relapsed patients studied at diagnosis [ 20 ]. CDK inhibitors are currently investigated as a promising therapeutic strategy either in newly diagnosed or in relapsed AML cases [ 42 , 43 , 44 ]. The high CSK2 kinase activity of relapsed patients at diagnosis was no longer observed in chemoresistant cells after relapse.…”

Highly Potent, Selective, and Orally Bioavailable 4-Thiazol-N-(pyridin-2-yl)pyrimidin-2-amine Cyclin-Dependent Kinases 4 and 6 Inhibitors as Anticancer Drug Candidates: Design, Synthesis, and Evaluation

“…Recently, we successfully optimized the pharmacokinetic (PK) properties of CDK9 inhibitors by introduction of a fluorine atom at C5 position of the pyrimidine ring. , This strategy was also deployed in the current study to seek potent and selective CDK4/6 inhibitors with improved bioavailability. In addition, we envisioned that the electronegative fluorine atom would acidify the bridging NH between the pyrimidine and pyridine rings, thereby increasing the strength of the hydrogen bonding with the carbonyl group of Val in the hinge region .…”

“…1 H NMR (500 MHz, CDCl 3 ) δ 2.35 (s, 3H), 2.56 (t, 4H, J 5.0), 3.45 (t, 4H, J 5.0), 7.18 (dd, 1H, J 9.0 and 3.0), 8.11 (d, 1H, J 3.0), 8.14 (d, 1H, J 9.5). 13 (37). Nitro compound 37 was prepared from homopiperazine 26 (5.03 g, 50.2 mmol) according to general synthetic procedure 4.…”

Targeting CDK9 for treatment of colorectal cancer

“…Recently, we demonstrated that CDKI-73 causes apoptosis through reduction of Mcl-1 and c-Myc in MLL-AML cell lines (Li et al, 2015). Mcl-1 and c-Myc are known to be regulated by CDK9-driven transcription and eIF4E-mediated translation (Hou et al, 2012;Wang and Fischer, 2008).…”